E. coli-based production of beta-lactamase

ABSTRACT

The invention relates to, in part, improved methods for the production of beta-lactamase using  Escherichia coli  ( E. coli ) cells. High yield production of beta-lactamase is achieved using methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage of International Patent ApplicationNo. PCT/US2015/47187, filed Aug. 27, 2015, which claims the benefit ofU.S. Provisional Patent Application No. 62/043,360, filed Aug. 28, 2014,the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to, in part, improved methods for the productionof beta-lactamases using Escherichia coli (E. coli) cells. High yieldproduction of beta-lactamase, including those suitable forpharmaceutical formulations, is achieved using methods of the invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:SYN-005PC-SequenceListing.txt; date recorded: Aug. 20, 2015; file size:19 KB).

BACKGROUND

Beta-lactam antibiotics are characterized by a beta-lactam ring in theirmolecular structure. The integrity of the beta-lactam ring is essentialfor the biological activity, which results in the inactivation of a setof transpeptidases that catalyze the final cross-linking reactions ofpeptidoglycan synthesis. Members of the beta-lactam antibiotics familyinclude penicillins, cephalosporins, clavams (or oxapenams), cephamycinsand carbapenems.

Beta-lactamases are bacterial defensive enzymes that hydrolyzebeta-lactam antibiotics. Gram-negative bacteria produce beta-lactamasesto achieve resistance to beta-lactam antibiotics. Particularly,beta-lactamases are able to efficiently catalyze the irreversiblehydrolysis of the amide bond of the beta-lactam ring resulting inbiologically inactive product(s).

Humans may be considered to be a “superorganism” which is a conglomerateof mammalian and microbial cells, with the latter estimated to outnumberthe former by ten to one. This microbial component, and its microbialgenetic repertoire, the microbiome, is roughly 100-times greater thanthat of the human host. Strikingly, despite this enormous diversity offoreign organisms, the human immune system generally maintains a stateof synergy. This is particularly true of the distal GI tract, whichhouses up to 1000 distinct bacterial species and an estimated excess of1×10¹⁴ microorganisms, and appears to be central in defining human hosthealth status. Loss of the careful balance in the microbiome, especiallyin the GI tract, can lead to various diseases.

Antibiotic medical treatments, which are needed to treat certain aspectsof disease, can induce disruption in the microbiome, including in the GItract, and lead to further disease. For instance, certain parentallyadministered beta-lactams like ampicillin, ceftriaxone, cefoperazone,and piperacillin are, in part, eliminated via biliary excretion into theproximal part of the small intestine (duodenum). Residual unabsorbedbeta-lactams in the intestinal tract may cause an undesirable effect onthe ecological balance of normal intestinal microbiota resulting in, forexample, Clostridium difficile infection (CDI), antibiotic-associateddiarrhea, overgrowth of pathogenic bacteria such as vancomycin resistantenterococci (VRE), extended-spectrum beta-lactamase producingGram-negative bacilli (ESBL), and fungi, and selection ofantibiotic-resistance strains among both normal intestinal microbiotaand potential pathogen bacteria.

One approach for avoiding or rebalancing the ecological balance ofnormal intestinal microbiota is the therapeutic use of beta-lactamases,for example, by inactivating excreted or unabsorbed antibiotics in theGI tract, thereby maintaining a normal intestinal microbiota andpreventing its overgrowth with potentially pathogenic micro-organisms.

Accordingly, there is remains a need for efficient methods of producingbeta-lactamases at a commercial scale for use in therapeuticintervention.

SUMMARY OF THE INVENTION

The present invention provides an improved method for the production ofa beta-lactamase polypeptide in Escherichia coli (E. coli) cells. Themethod includes providing a host E. coli cell transformed with a vectorcomprising a sequence encoding the beta-lactamase polypeptide. The E.coli cell is cultured to induce expression of the beta-lactamase in thecytoplasm. Soluble fractions are subsequently prepared from the E. colicell to recover the beta-lactase polypeptide.

The methods of the invention allows for production of beta-lactamases ata high yield. In an embodiment, the method yields at least 10 grams ofthe beta-lactamase polypeptide per liter of culture. In anotherembodiment, the method yields at least 15 grams of the beta-lactamasepolypeptide per liter of culture.

Various strains of E. coli cells may be employed for the instantinvention. For example, the E. coli cell may be selected from BL21 (DE3)or W3110. The beta-lactamase polypeptide is predominantly expressed inthe cytoplasm of the E. coli cell. In certain embodiments, expression ofthe polypeptide is induced by adding isopropylthiogalactoside (IPTG) tothe culture.

The disclosed method may be utilized to produce beta-lactamases andderivatives thereof. In one embodiment, the beta-lactamase polypeptidecomprises a sequence having at least 60% identity with P1A. In anotherembodiment, the beta-lactamase polypeptide comprises a sequence havingat least 60% identity with P2A. In yet another embodiment, thebeta-lactamase polypeptide comprises a sequence having at least 60%identity with P3A. In a further embodiment, the beta-lactamasepolypeptide comprises a sequence having at least 60% identity with P4A.In various embodiments, the present methods are used to producebeta-lactamases useful for microbiome-protecting therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a multi-fermenter computer system (MFCS) CLD977fermentation plot of batch age (hours) vs. airflow (AIRFL (l/min),second line from top), temperature (TEMP (° C.), top line), stirringrate (STIRR (RPM), second line from the bottom), pH (third line), andpercent oxygen (PO₂, bottom line).

FIG. 2 shows a MFCS CLD990 fermentation plot of batch age (hours) vs.airflow (AIRFL (l/min), second line from top), temperature (TEMP (° C.),top line), stirring rate (STIRR (RPM), second line from the bottom), pH(third line), and percent oxygen (PO₂, bottom line).

FIG. 3 shows a MFCS fermentation exit gas analysis plot of batch age(hours) vs. CLD977 (3/13C039) and CLD990 (4/13C040) oxygen uptake rate(OUR) and carbon dioxide evolution rate (CER) (mM/l/hr). Labeled fromleft to right, the first line corresponds to CLD977 OUR, the second linecorresponds to CLD977 CER, the third line corresponds to CLD990 OUR andthe fourth line corresponds to CLD990 CER.

FIG. 4 shows a biomass plot for CLD977 (3/13C039) and CLD990 (4/13C040)of batch time (hours) vs. OD₆₀₀ and dry cell weight (DCW (g/L)). CLD977OD₆₀₀ and DCW lines correspond to the top line and second from bottomline, respectively. CLD990 OD₆₀₀ and DCW lines correspond to the secondfrom top line and bottom line, respectively.

FIG. 5 shows bacterial gram stains for CLD977 and CLD990 at the end ofbatch phase and after fermentation is complete (final sample).

FIG. 6 shows SDS PAGE analysis of CLD977 (3/13CO39) time course samplesfrom pre-induction to the end of fermentation compared to controlstandards.

FIG. 7 shows SDS PAGE analysis of CLD990 (4/13C040) time course samplesfrom pre-induction to the end of fermentation compared to controlstandards.

FIG. 8 shows SDS PAGE analysis of sonicated samples from CLD977(3/13C039) and CLD990 (4/13C040) compared to control standards. CLD977and CLD990 yielded mostly soluble protein. Only faint product bands areseen for the insoluble fraction.

FIG. 9 shows a standard curve of Time (sec) vs. Absorbance for Controls1 and 2 as well as reference standard dilutions of 0.6, 0.8, 1.0, 1.5,2.0, and 4 mg/L. Controls 1 and 2 were preset dilutions of 1.0 μg/mL ranas duplicates.

FIG. 10 shows a standard end point curve of Standard Concentration(mg/L) vs. End Point Absorbance. Standard absorbance was measured attime=60 sec minus standard absorbance at time=0 sec. Specifically,enzymatic reaction was measured at time=60 sec. The absorbance wasmeasured at time=0 sec which was then subtracted from the 60 secmeasurement. Several dilutions of the reference standard were tested togenerate a standard curve.

FIG. 11 shows a standard curve of Time (sec) vs. Absorbance for CLD981(3/13C037 (also referred to as 37)) at 12 hours, 24 hours, 48 hours, aswell as the periplasmic osmotic shock fraction (OS2). Specifically, OS2is the second buffer fraction prepared from an E. coli pellet andrepresents the periplasmic space fraction.

FIG. 12 shows a standard end point curve of Time (sec) vs. Absorbancefor CLD981 (3/13C037) OS1 samples.

FIG. 13 shows a standard curve of Time (sec) vs. Absorbance for CLD982(4/13C038 (also referred to as 38)) 12 h, 24 h, 48 h, and OS1 and OS2 48h post induction.

FIG. 14 shows a standard curve of Time (sec) vs. Absorbance for Control1 and 2 (combined into control standard) as well as reference standardmaterial dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L.

FIG. 15 shows a standard end point curve of Standard Concentration(mg/L) vs. End Point Absorbance. Standard absorbance was measured attime=60 sec minus standard absorbance at time=0 sec.

FIG. 16 shows a standard curve of Time (sec) vs. Absorbance for CLD981(37) and CLD982 (38) OS1 and OS2 48 h post induction. Table 3 is asummary of assay plate 2 activity and titer results for CLD981 andCLD982 OS1 and OS2 along with controls 1 and 2.

FIG. 17 shows a standard curve of Time (sec) vs. Absorbance for Control1 and 2 (combined as control standard) as well as reference standardmaterial dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L.

FIG. 18 shows a standard end point curve of Standard Concentration(mg/L) vs. End Point Absorbance. Standard absorbance was measured attime=60 sec minus standard absorbance at time=0 sec.

FIG. 19 shows a standard curve of Time (sec) vs. Absorbance for CLD977(39) and CLD 990 (40) for both the second to last and last time pointpost induction (unlabeled=sonication) as well as the last time pointpost induction (Bug buster).

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discoverythat a beta-lactamase polypeptide can be overproduced in high yields inE coli cells. Specifically, high yield production is achieved byexpressing the polypeptide in the cytoplasm of E coli cells andsubsequently recovering the polypeptide from soluble fractions preparedfrom the cells.

Prior to the present invention, it was well established thatbeta-lactamases, such as the beta-lactamase from Bacillus licheniformis,are mostly found in the cell envelope and periplasmic fractions of Ecoli cells. See Mezes, et al., J Biol Chem (1983), 258(18): 11211-11218.Particularly, beta-lactamase from Bacillus licheniformis is found to becompletely absent in the cytoplasm. Id.

Further still, production of beta-lactamases from E coli cells hasgenerally been inefficient leading to an overall yield on the scale ofmilligrams of the enzyme per liter of culture. See, for example, Shaw etal., Protein Expr Purif. (1991), 2(2-3): 151-157. Given that thebeta-lactamases are from Bacillus licheniformis, it is expected thatproduction of these enzymes in Bacillus strains may provide a higheryield. However, studies shown herein demonstrate that even when producedin Bacillus subtilis cells, the yield of beta-lactamases is low.Accordingly, it is surprising that the present invention achieves anoverall yield of beta-lactamases on the scale of grams per liter ofculture.

Accordingly, the present invention provides an improved method for theproduction of a beta-lactamase polypeptide in Escherichia coli (E. coli)cells. The method includes providing a host E. coli cell transformedwith a vector comprising a sequence encoding the beta-lactamasepolypeptide. The E. coli cell is cultured to induce expression of thebeta-lactamase in the cytoplasm. Soluble fractions are subsequentlyprepared from the E. coli cell for recovery of the beta-lactasepolypeptide.

The present invention allows for high-yield production of abeta-lactamase polypeptide in E coli cells. In various embodiments,methods of the present invention provides a yield of at least about 1gram, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about6 grams, about 7 grams, about 8 grams, about 9 grams, about 10 grams,about 11 grams, about 12 grams, about 13 grams, about 14 grams, about 15grams, about 16 grams, about 17 grams, about 18 grams, about 19 grams,about 20 grams, about 22 grams, about 24 grams, about 26 grams, about 28grams, about 30 grams, about 35 grams, about 40 grams, about 45 grams,or about 50 grams of the beta-lactamase polypeptide per liter ofculture. In one embodiment, at least about 10 grams of the beta-lactasepolypeptide per liter of culture is recovered. In another embodiment,about at least 15 grams of the beta-lactase polypeptide per liter ofculture is recovered. In a further embodiment, at least about 18 gramsof the beta-lactase polypeptide per liter of culture is recovered.

In various embodiments, the present methods provide one or more ofgreater yield and improved purity as compared to a Bacillus-basedexpression system such as, for example, those described in U.S. Pat. No.7,319,030, the entire contents of which are hereby incorporated byreference. In various embodiments, the present methods provide one ormore of greater yield and improved purity as compared to a method forproducing a desired polypeptide product using a non-sporulating Bacillussubtilis strain, in which a deletion region of at least 150 nucleotideshas been deleted from its sigG gene, the method involving transformingthe strain with a polynucleotide construct encoding a recombinantpolypeptide, expressing the polynucleotide construct, and recovering therecombinant polypeptide. In some embodiments the method comprisesdeleting at least part of either of the two functional regions of thesigG gene (i.e. the regions which code for amino acids 67 to 80 or 229to 248).

In various embodiments, the present methods provide about a 5-fold, orabout a 7.5-fold, or about a 10-fold, or about a 15-fold improvement inyield in E. coli versus a Bacillus-based expression system such as, forexample, those described in U.S. Pat. No. 7,319,030.

Various E. coli cell can be used with the present invention.Illustrative E. coli cells include, but are not limited to, BL21 (DES),W3110, DH5α, HMS174, and derivatives thereof. In one embodiment, the E.coli cell is the BL21 (DES) strain. In another embodiment, the E. colicell is W3110 strain. The genotype of W3110 is E coli K12 F-, λ-, IN(rrnD-rrnE)1, rph-1. It is a Gram negative, rod-shaped, facultativeanaerobe, and its genealogy is well described (Bachmann, B J 1972.Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol.Rev. 36(4):525-57). There have been no modifications of this strainprior to transformation with the B3214 plasmid.

The present invention is used to produce beta-lactamase polypeptides ata high yield. In various aspects, the beta-lactamases polypeptide hasthe sequence of SEQ ID NO: 1 (Bacillus licheniformis PenP, i.e., P1A) oris derived by one or more mutations of SEQ ID NO: 1. Provided herein isthe 263 amino acid sequence of the P1A enzyme (after removal of a 31amino acid signal sequence and the QASKT (Gln-Ala-Ser-Lys-Thr) (SEQ IDNO: 11) pentapeptide at the N terminus, see SEQ ID NO: 3). As describedherein, mutations may be made to this sequence to generatebeta-lactamase derivatives that may be produced by methods of theinvention.

SEQ ID NO: 1 EMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVADKTGAASYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDDKLIAEATKVVMKALNMNGK.

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises an amino acid sequence having at least about60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%) sequence identity with SEQ ID NO: 1.

In some embodiments, SEQ ID NO: 1 may have a Met and/or Thr precedingthe first residue of the sequence. In various embodiments, the Met maybe cleaved. As described herein, mutations may be made to the sequencecomprising the Met and/or Thr preceding the first residue to generatebeta-lactamase derivatives.

Also provided herein is the 299 amino acid sequence of the P1A enzymebefore removal of a 31 amino acid signal sequence and the QASKT(Gln-Ala-Ser-Lys-Thr) (SEQ ID NO: 11) pentapeptide at the N terminus asSEQ ID NO: 3:

SEQ ID NO: 3 MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAQASKTEMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVADKTGAASYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDDKLIAEATK VVMKALNMNGK

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises an amino acid sequence having at least about60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%) sequence identity with SEQ ID NO: 3.

Further, the beta-lactamase polypeptide may include additional upstreamresidues from the first residue of SEQ ID NO: 1 (see, e.g., JBC 258(18): 11211, 1983, the contents of which are hereby incorporated byreference-including the exo-large and exo-small versions of penP andpenP1). Further, the beta-lactamase polypeptide may also includeadditional downstream residues from the last residue of SEQ ID NO: 1.

The polynucleotide sequence of P1A (after removal of a 31 amino acidsignal sequence and the QAKST (SEQ ID NO: 12) pentapeptide at the Nterminus) is provided as SEQ ID NO: 2. As described herein, mutationsmay be made to this sequence to generate the beta-lactamase derivatives(including, taking into account degeneracy of the genetic code).

SEQ ID NO: 2 gagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, the polynucleotide of the present invention has atleast about 60% (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99%) sequence identity with SEQ ID NO: 2.

Also provided is the polynucleotide sequence of P1A before the removalof a 31 amino acid signal sequence and the QASKT (SEQ ID NO: 11)pentapeptide at the N terminus as SEQ ID NO: 4. As described herein,mutations may be made to this sequence to generate beta-lactamasederivatives (including, taking into account degeneracy of the geneticcode).

SEQ ID NO: 4 atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagcgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, the polynucleotide of the present invention has atleast about 60% (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99%) sequence identity with SEQ ID NO: 4.

In some embodiments, mutagenesis of a beta-lactamase (e.g. a class Abeta-lactamase) is performed to derive advantageous enzymes (e.g. thosethat can target a broad spectra of antibiotics). In some embodiments,beta-lactamases derivatives are obtained by site-directed mutagenesis,random mutagenesis, and/or directed evolution approaches. In someembodiments, mutation design is based on, inter alia, structural data(e.g. crystal structure data, homolog models, etc.) of the following:P1A crystal structure (Knox and Moews, J. Mol Biol., 220, 435-455(1991)), CTX-M-44 (1BZA (Ibuka et al. Journal of Molecular BiologyVolume 285, Issue 5 2079-2087 (1999), 1IYS (Ibuka et al. Biochemistry,2003, 42 (36): 10634-43), 1IYO, 1IYP and 1IYQ (Shimamura et al. 2002 J.Biol. Chem. 277:46601-08), Proteus vulgaris K1 (1HZO, Nugaka et al. JMol Biol. 2002 Mar. 15; 317(1):109-17) and Proteus penneri HugA(Liassine et al. Antimicrob Agents Chemother. 2002 January; 46(1):216-9.2002), and reviewed in Bonnet, Antimicrob. Agents Chemother 48(1): 1-14(2004) (for CTM-X), the contents of all of these documents are herebyincorporated by reference in their entirety). In some embodiments, thepresent mutations are informed by analysis of structural data (e.g.crystal structure data, homolog models, etc.) of any one of thefollowing beta-lactamases: P1A (see, e.g. U.S. Pat. No. 5,607,671, thecontents of which are hereby incorporated by reference), P2A (see, e.g.,WO 2007/147945, the contents of which are hereby incorporated byreference), P3A (see, e.g., WO 2011/148041, the contents of which arehereby incorporated by reference), CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-9,CTX-M-10, CTX-M-14, CTX-M-15, CTX-M-16, CTX-M-18, CTX-M-19, CTX-M-25,CTX-M-26, CTX-M-27, CTX-M-32, CTX-M-44, CTX-M-45, and CTX-M-54. Suchinformation is available to one skilled in the art at known databases,for example, Swiss-Prot Protein Sequence Data Bank, NCBI, and PDB.

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention includes one or more (e.g. about 1, or about 2, orabout 3, or about 4, or about 5, or about 6, or about 7, or about 8, orabout 9, or about 10, or about 15, or about 20, or about 30, or about40, or about 50, or about 60, or about 70, or about 80, or about 90, orabout 100, or about 110, or about 120, or about 130, or about 140, orabout 150) mutations relative to SEQ ID NO: 1 or SEQ ID NO: 3 or asequence with at least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity to SEQ ID NO: 1 or SEQ IDNO: 3 (or about 60%, about 65%, about 70%, or about 75%, or about 80%,or about 85%, or about 90, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 3). Invarious embodiments, one or more amino acid of SEQ ID NO: 1 or SEQ IDNO: 3 is substituted with a naturally occurring amino acid, such as ahydrophilic amino acid (e.g. a polar and positively charged hydrophilicamino acid, such as arginine (R) or lysine (K); a polar and neutral ofcharge hydrophilic amino acid, such as asparagine (N), glutamine (Q),serine (S), threonine (T), proline (P), and cysteine (C), a polar andnegatively charged hydrophilic amino acid, such as aspartate (D) orglutamate (E), or an aromatic, polar and positively charged hydrophilicamino acid, such as histidine (H)) or a hydrophobic amino acid (e.g. ahydrophobic, aliphatic amino acid such as glycine (G), alanine (A),leucine (L), isoleucine (I), methionine (M), or valine (V), ahydrophobic, aromatic amino acid, such as phenylalanine (F), tryptophan(W), or tyrosine (Y) or a non-classical amino acid (e.g. selenocysteine,pyrrolysine, N-formylmethionine β-alanine, GABA and δ-Aminolevulinicacid. 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteicacid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β methylamino acids, C α-methyl amino acids, N α-methyl amino acids, and aminoacid analogs in general).

In illustrative embodiments, inventive mutations include, but are notlimited to one or more (e.g. about 1, or about 2, or about 3, or about4, or about 5, or about 6, or about 7, or about 8, or about 9, or about10, or about 15, or about 20, or about 30, or about 40, or about 50, orabout 60, or about 70, or about 80, or about 90, or about 100, or about110, or about 120, or about 130, or about 140, or about 150) of thefollowing mutations to SEQ ID NO: 1 or SEQ ID NO: 3 or a sequence withat least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 99.5, 99.8, 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 3 (or about70%, or about 75%, or about 80%, or about 85%, or about 90, or about95%, or about 96%, or about 97%, or about 98%, or about 99% identity toSEQ ID NO: 1 or SEQ ID NO: 3): Glu1Ala; Glu1Cys; Glu1Asp; Glu1Phe;Glu1Gly; Glu1 His; Glu1 Ile; Met1Lys; Glu1Leu; Glu1Met; Glu1Asn;Glu1Pro; Glu1Gln; Glu1Arg; Glu1Ser; Glu1Thr; Glu1Val; Glu1Trp; Glu1Tyr;Met2Ala; Met2Cys; Met2Asp; Met2Glu; Met2Phe; Met2Gly; Met2His; Met2Ile;Met1 Lys; Met2Leu; Met2Asn; Met2Pro; Met2Gln; Met2Arg; Met2Ser; Met2Thr;Met2Val; Met2Trp; Met2Tyr; Lys3Ala; Lys3Cys; Lys3Asp; Lys3Glu; Lys3Phe;Lys3Gly; Lys3His; Lys3Ile; Lys3Leu; Lys3Met; Lys3Asn; Lys3Pro; Lys3Gln;Lys3Arg; Lys3Ser; Lys3Thr; Lys3Val; Lys3Trp; Lys3Tyr; Asp4Ala; Asp4Cys;Asp4Glu; Asp4Phe; Asp4Gly; Asp4His; Asp4Ile; Asp4Lys; Asp4Leu; Asp4Met;Asp4Asn; Asp4Pro; Asp4Gln; Asp4Arg; Asp4Ser; Asp4Thr; Asp4Val; Asp4Trp;Asp4Tyr; Asp5Ala; Asp5Cys; Asp5Glu; Asp5Phe; Asp5Gly; Asp5His; Asp5Ile;Asp5Lys; Asp5Leu; Asp5Met; Asp5Asn; Asp5Pro; Asp5Gln; Asp5Arg; Asp5Ser;Asp5Thr; Asp5Val; Asp5Trp; Asp5Tyr; Phe6Ala; Phe6Cys; Phe6Asp; Phe6Glu;Phe6Gly; Phe6His; Phe6Ile; Phe6Lys; Phe6Leu; Phe6Met; Phe6Asn; Phe6Pro;Phe6Gln; Phe6Arg; Phe6Ser; Phe6Thr; Phe6Val; Phe6Trp; Phe6Tyr; Ala7Cys;Ala7Asp; Ala7Glu; Ala7Phe; Ala7Gly; Ala7His; Ala7Ile; Ala7Lys; Ala7Leu;Ala7Met; Ala7Asn; Ala7Pro; Ala7Gln; Ala7Arg; Ala7Ser; Ala7Thr; Ala7Val;Ala7Trp; Ala7Tyr; Lys8Ala; Lys8Cys; Lys8Asp; Lys8Glu; Lys8Phe; Lys8Gly;Lys8His; Lys8Ile; Lys8Leu; Lys8Met; Lys8Asn; Lys8Pro; Lys8Gln; Lys8Arg;Lys8Ser; Lys8Thr; Lys8Val; Lys8Trp; Lys8Tyr; Leu9Ala; Leu9Cys; Leu9Asp;Leu9Glu; Leu9Phe; Leu9Gly; Leu9His; Leu9Ile; Leu9Lys; Leu9Met; Leu9Asn;Leu9Pro; Leu9Gln; Leu9Arg; Leu9Ser; Leu9Thr; Leu9Val; Leu9Trp; Leu9Tyr;Glu10Ala; Glu10Cys; Glu10Asp; Glu10Phe; Glu10Gly; Glu10His; Glu10Ile;Glu10Lys; Glu10Leu; Glu10Met; Glu10Asn; Glu10Pro; Glu10Gln; Glu10Arg;Glu10Ser; Glu10Thr; Glu10Val; Glu10Trp; Glu10Tyr; Glu11Ala; Glu11Cys;Glu11Asp; Glu11Phe; Glu11Gly; Glu11His; Glu11Ile; Glu11Lys; Glu11Leu;Glu11Met; Glu11Asn; Glu11Pro; Glu11Gln; Glu11Arg; Glu11Ser; Glu11Thr;Glu11Val; Glu11Trp; Glu11Tyr; Gln12Ala; Gln12Cys; Gln12Asp; Gln12Glu;Gln12Phe; Gln12Gly; Gln12His; Gln12Ile; Gln12Lys; Gln12Leu; Gln12Met;Gln12Asn; Gln12Pro; Gln12Arg; Gln12Ser; Gln12Thr; Gln12Val; Gln12Trp;Gln12Tyr; Phe13Ala; Phe13Cys; Phe13Asp; Phe13Glu; Phe13Gly; Phe13His;Phe13Ile; Phe13Lys; Phe13Leu; Phe13Met; Phe13Asn; Phe13Pro; Phe13Gln;Phe13Arg; Phe13Ser; Phe13Thr; Phe13Val; Phe13Trp; Phe13Tyr; Asp14Ala;Asp14Cys; Asp14Glu; Asp14Phe; Asp14Gly; Asp14His; Asp14Ile; Asp14Lys;Asp14Leu; Asp14Met; Asp14Asn; Asp14Pro; Asp14Gln; Asp14Arg; Asp14Ser;Asp14Thr; Asp14Val; Asp14Trp; Asp14Tyr; Ala15Cys; Ala15Asp; Ala15Glu;Ala15Phe; Ala15Gly; Ala15His; Ala15Ile; Ala15Lys; Ala15Leu; Ala15Met;Ala15Asn; Ala15Pro; Ala15Gln; Ala15Arg; Ala15Ser; Ala15Thr; Ala15Val;Ala15Trp; Ala15Tyr; Lys16Ala; Lys16Cys; Lys16Asp; Lys16Glu; Lys16Phe;Lys16Gly; Lys16His; Lys16Ile; Lys16Leu; Lys16Met; Lys16Asn; Lys16Pro;Lys16Gln; Lys16Arg; Lys16Ser; Lys16Thr; Lys16Val; Lys16Trp; Lys16Tyr;Leu17Ala; Leu17Cys; Leu17Asp; Leu17Glu; Leu17Phe; Leu17Gly; Leu17His;Leu17Ile; Leu17Lys; Leu17Met; Leu17Asn; Leu17Pro; Leu17Gln; Leu17Arg;Leu17Ser; Leu17Thr; Leu17Val; Leu17Trp; Leu17Tyr; Gly18Ala; Gly18Cys;Gly18Asp; Gly18Glu; Gly18Phe; Gly18His; Gly18Ile; Gly18Lys; Gly18Leu;Gly18Met; Gly18Asn; Gly18Pro; Gly18Gln; Gly18Arg; Gly18Ser; Gly18Thr;Gly18Val; Gly18Trp; Gly18Tyr; Ile19Ala; Ile19Cys; Ile19Asp; Ile19Glu;Ile19Phe; Ile19Gly; Ile19His; Ile19Lys; Ile19Leu; Ile19Met; Ile19Asn;Ile19Pro; Ile19Gln; Ile19Arg; Ile19Ser; Ile19Thr; Ile19Val; Ile19Trp;Ile19Tyr; Phe20Ala; Phe20Cys; Phe20Asp; Phe20Glu; Phe20Gly; Phe20His;Phe20Ile; Phe20Lys; Phe20Leu; Phe20Met; Phe20Asn; Phe20Pro; Phe20Gln;Phe20Arg; Phe20Ser; Phe20Thr; Phe20Val; Phe20Trp; Phe20Tyr; Ala21Cys;Ala21Asp; Ala21Glu; Ala21Phe; Ala21Gly; Ala21His; Ala21Ile; Ala21Lys;Ala21Leu; Ala21Met; Ala21Asn; Ala21Pro; Ala21Gln; Ala21Arg; Ala21Ser;Ala21Thr; Ala21Val; Ala21Trp; Ala21Tyr; Leu22Ala; Leu22Cys; Leu22Asp;Leu22Glu; Leu22Phe; Leu22Gly; Leu22His; Leu22Ile; Leu22Lys; Leu22Met;Leu22Asn; Leu22Pro; Leu22Gln; Leu22Arg; Leu22Ser; Leu22Thr; Leu22Val;Leu22Trp; Leu22Tyr; Asp23Ala; Asp23Cys; Asp23Glu; Asp23Phe; Asp23Gly;Asp23His; Asp23Ile; Asp23Lys; Asp23Leu; Asp23Met; Asp23Asn; Asp23Pro;Asp23Gln; Asp23Arg; Asp23Ser; Asp23Thr; Asp23Val; Asp23Trp; Asp23Tyr;Thr24Ala; Thr24Cys; Thr24Asp; Thr24Glu; Thr24Phe; Thr24Gly; Thr24His;Thr24Ile; Thr24Lys; Thr24Leu; Thr24Met; Thr24Asn; Thr24Pro; Thr24Gln;Thr24Arg; Thr24Ser; Thr24Val; Thr24Trp; Thr24Tyr; Gly25Ala; Gly25Cys;Gly25Asp; Gly25Glu; Gly25Phe; Gly25His; Gly25Ile; Gly25Lys; Gly25Leu;Gly25Met; Gly25Asn; Gly25Pro; Gly25Gln; Gly25Arg; Gly25Ser; Gly25Thr;Gly25Val; Gly25Trp; Gly25Tyr; Thr26Ala; Thr26Cys; Thr26Asp; Thr26Glu;Thr26Phe; Thr26Gly; Thr26His; Thr26Ile; Thr26Lys; Thr26Leu; Thr26Met;Thr26Asn; Thr26Pro; Thr26Gln; Thr26Arg; Thr26Ser; Thr26Val; Thr26Trp;Thr26Tyr; Asn27Ala; Asn27Cys; Asn27Asp; Asn27Glu; Asn27Phe; Asn27Gly;Asn27His; Asn27Ile; Asn27Lys; Asn27Leu; Asn27Met; Asn27Pro; Asn27Gln;Asn27Arg; Asn27Ser; Asn27Thr; Asn27Val; Asn27Trp; Asn27Tyr; Arg28Ala;Arg28Cys; Arg28Asp; Arg28Glu; Arg28Phe; Arg28Gly; Arg28His; Arg28Ile;Arg28Lys; Arg28Leu; Arg28Met; Arg28Asn; Arg28Pro; Arg28Gln; Arg28Ser;Arg28Thr; Arg28Val; Arg28Trp; Arg28Tyr; Thr29Ala; Thr29Cys; Thr29Asp;Thr29Glu; Thr29Phe; Thr29Gly; Thr29His; Thr29Ile; Thr29Lys; Thr29Leu;Thr29Met; Thr29Asn; Thr29Pro; Thr29Gln; Thr29Arg; Thr29Ser; Thr29Val;Thr29Trp; Thr29Tyr; Val30Ala; Val30Cys; Val30Asp; Val30Glu; Val30Phe;Val30Gly; Val30His; Val30Ile; Val30Lys; Val30Leu; Val30Met; Val30Asn;Val30Pro; Val30Gln; Val30Arg; Val30Ser; Val30Thr; Val30Trp; Val30Tyr;Ala31Ala; Ala31Cys; Ala31Asp; Ala31Glu; Ala31Phe; Ala31Gly; Ala31His;Ala31Ile; Ala31Lys; Ala31Leu; Ala31Met; Ala31Asn; Ala31Pro; Ala31Gln;Ala31Arg; Ala31Ser; Ala31Thr; Ala31Val; Ala31Trp; Ala31Tyr; Tyr32Ala;Tyr32Cys; Tyr32Asp; Tyr32Glu; Tyr32Phe; Tyr32Gly; Tyr32His; Tyr32Ile;Tyr32Lys; Tyr32Leu; Tyr32Met; Tyr32Asn; Tyr32Pro; Tyr32Gln; Tyr32Arg;Tyr32Ser; Tyr32Thr; Tyr32Val; Tyr32Trp; Arg33Ala; Arg33Cys; Arg33Asp;Arg33Glu; Arg33Phe; Arg33Gly; Arg33His; Arg33Ile; Arg33Lys; Arg33Leu;Arg33Met; Arg33Asn; Arg33Pro; Arg33Gln; Arg33Ser; Arg33Thr; Arg33Val;Arg33Trp; Arg33Tyr; Pro34Ala; Pro34Cys; Pro34Asp; Pro34Glu; Pro34Phe;Pro34Gly; Pro34His; Pro34Ile; Pro34Lys; Pro34Leu; Pro34Met; Pro34Asn;Pro34Gln; Pro34Arg; Pro34Ser; Pro34Thr; Pro34Val; Pro34Trp; Pro34Tyr;Asp35Ala; Asp35Cys; Asp35Glu; Asp35Phe; Asp35Gly; Asp35His; Asp35Ile;Asp35Lys; Asp35Leu; Asp35Met; Asp35Asn; Asp35Pro; Asp35Gln; Asp35Arg;Asp35Ser; Asp35Thr; Asp35Val; Asp35Trp; Asp35Tyr; Glu36Ala; Glu36Cys;Glu36Asp; Glu36Phe; Glu36Gly; Glu36His; Glu36Ile; Glu36Lys; Glu36Leu;Glu36Met; Glu36Asn; Glu36Pro; Glu36Gln; Glu36Arg; Glu36Ser; Glu36Thr;Glu36Val; Glu36Trp; Glu36Tyr; Arg37Ala; Arg37Cys; Arg37Asp; Arg37Glu;Arg37Phe; Arg37Gly; Arg37His; Arg37Ile; Arg37Lys; Arg37Leu; Arg37Met;Arg37Asn; Arg37Pro; Arg37Gln; Arg37Ser; Arg37Thr; Arg37Val; Arg37Trp;Arg37Tyr; Phe38Ala; Phe38Cys; Phe38Asp; Phe38Glu; Phe38Gly; Phe38His;Phe38Ile; Phe38Lys; Phe38Leu; Phe38Met; Phe38Asn; Phe38Pro; Phe38Gln;Phe38Arg; Phe38Ser; Phe38Thr; Phe38Val; Phe38Trp; Phe38Tyr; Ala39Cys;Ala39Asp; Ala39Glu; Ala39Phe; Ala39Gly; Ala39His; Ala39Ile; Ala39Lys;Ala39Leu; Ala39Met; Ala39Asn; Ala39Pro; Ala39Gln; Ala39Arg; Ala39Ser;Ala39Thr; Ala39Val; Ala39Trp; Ala39Tyr; Phe40Ala; Phe40Cys; Phe40Asp;Phe40Glu; Phe40Gly; Phe40His; Phe40Ile; Phe40Lys; Phe40Leu; Phe40Met;Phe40Asn; Phe40Pro; Phe40Gln; Phe40Arg; Phe40Ser; Phe40Thr; Phe40Val;Phe40Trp; Phe40Tyr; Ala41Cys; Ala41Asp; Ala41Glu; Ala41Phe; Ala41Gly;Ala41His; Ala41Ile; Ala41Lys; Ala41Leu; Ala41Met; Ala41Asn; Ala41Pro;Ala41Gln; Ala41Arg; Ala41Ser; Ala41Thr; Ala41Val; Ala41Trp; Ala41Tyr;Ser42Ala; Ser42Cys; Ser42Asp; Ser42Glu; Ser42Phe; Ser42Gly; Ser42His;Ser42Ile; Ser42Lys; Ser42Leu; Ser42Met; Ser42Asn; Ser42Pro; Ser42Gln;Ser42Arg; Ser42Thr; Ser42Val; Ser42Trp; Ser42Tyr; Thr43Ala; Thr43Cys;Thr43Asp; Thr43Glu; Thr43Phe; Thr43Gly; Thr43His; Thr43Ile; Thr43Lys;Thr43Leu; Thr43Met; Thr43Asn; Thr43Pro; Thr43Gln; Thr43Arg; Thr43Ser;Thr43Val; Thr43Trp; Thr43Tyr; Ile44Ala; Ile44Cys; Ile44Asp; Ile44Glu;Ile44Phe; Ile44Gly; Ile44His; Ile44Lys; Ile44Leu; Ile44Met; Ile44Asn;Ile44Pro; Ile44Gln; Ile44Arg; Ile44Ser; Ile44Thr; Ile44Val; Ile44Trp;Ile44Tyr; Lys45Ala; Lys45Cys; Lys45Asp; Lys45Glu; Lys45Phe; Lys45Gly;Lys45His; Lys45Ile; Lys45Leu; Lys45Met; Lys45Asn; Lys45Pro; Lys45Gln;Lys45Arg; Lys45Ser; Lys45Thr; Lys45Val; Lys45Trp; Lys45Tyr; Ala46Cys;Ala46Asp; Ala46Glu; Ala46Phe; Ala46Gly; Ala46His; Ala46Ile; Ala46Lys;Ala46Leu; Ala46Met; Ala46Asn; Ala46Pro; Ala46Gln; Ala46Arg; Ala46Ser;Ala46Thr; Ala46Val; Ala46Trp; Ala46Tyr; Leu47Ala; Leu47Cys; Leu47Asp;Leu47Glu; Leu47Phe; Leu47Gly; Leu47His; Leu47Ile; Leu47Lys; Leu47Met;Leu47Asn; Leu47Pro; Leu47Gln; Leu47Arg; Leu47Ser; Leu47Thr; Leu47Val;Leu47Trp; Leu47Tyr; Thr48Ala; Thr48Cys; Thr48Asp; Thr48Glu; Thr48Phe;Thr48Gly; Thr48His; Thr48Ile; Thr48Lys; Thr48Leu; Thr48Met; Thr48Asn;Thr48Pro; Thr48Gln; Thr48Arg; Thr48Ser; Thr48Val; Thr48Trp; Thr48Tyr;Val49Ala; Val49Cys; Val49Asp; Val49Glu; Val49Phe; Val49Gly; Val49His;Val49Ile; Val49Lys; Val49Leu; Val49Met; Val49Asn; Val49Pro; Val49Gln;Val49Arg; Val49Ser; Val49Thr; Val49Trp; Val49Tyr; Gly50Ala; Gly50Cys;Gly50Asp; Gly50Glu; Gly50Phe; Gly50His; Gly50Ile; Gly50Lys; Gly50Leu;Gly50Met; Gly50Asn; Gly50Pro; Gly50Gln; Gly50Arg; Gly50Ser; Gly50Thr;Gly50Val; Gly50Trp; Gly50Tyr; Val51Ala; Val51Cys; Val51Asp; Val51Glu;Val51Phe; Val51Gly; Val51His; Val51Ile; Val51Lys; Val51Leu; Val51Met;Val51Asn; Val51Pro; Val51Gln; Val51Arg; Val51Ser; Val51Thr; Val51Trp;Val51Tyr; Leu52Ala; Leu52Cys; Leu52Asp; Leu52Glu; Leu52Phe; Leu52Gly;Leu52His; Leu52Ile; Leu52Lys; Leu52Met; Leu52Asn; Leu52Pro; Leu52Gln;Leu52Arg; Leu52Ser; Leu52Thr; Leu52Val; Leu52Trp; Leu52Tyr; Leu53Ala;Leu53Cys; Leu53Asp; Leu53Glu; Leu53Phe; Leu53Gly; Leu53His; Leu53Ile;Leu53Lys; Leu53Met; Leu53Asn; Leu53Pro; Leu53Gln; Leu53Arg; Leu53Ser;Leu53Thr; Leu53Val; Leu53Trp; Leu53Tyr; Gln54Ala; Gln54Cys; Gln54Asp;Gln54Glu; Gln54Phe; Gln54Gly; Gln54His; Gln54Ile; Gln54Lys; Gln54Leu;Gln54Met; Gln54Asn; Gln54Pro; Gln54Arg; Gln54Ser; Gln54Thr; Gln54Val;Gln54Trp; Gln54Tyr; Gln55Ala; Gln55Cys; Gln55Asp; Gln55Glu; Gln55Phe;Gln55Gly; Gln55His; Gln55Ile; Gln55Lys; Gln55Leu; Gln55Met; Gln55Asn;Gln55Pro; Gln55Arg; Gln55Ser; Gln55Thr; Gln55Val; Gln55Trp; Gln55Tyr;Lys56Ala; Lys56Cys; Lys56Asp; Lys56Glu; Lys56Phe; Lys56Gly; Lys56His;Lys56Ile; Lys56Leu; Lys56Met; Lys56Asn; Lys56Pro; Lys56Gln; Lys56Arg;Lys56Ser; Lys56Thr; Lys56Val; Lys56Trp; Lys56Tyr; Ser57Ala; Ser57Cys;Ser57Asp; Ser57Glu; Ser57Phe; Ser57Gly; Ser57His; Ser57Ile; Ser57Lys;Ser57Leu; Ser57Met; Ser57Asn; Ser57Pro; Ser57Gln; Ser57Arg; Ser57Thr;Ser57Val; Ser57Trp; Ser57Tyr; Ile58Ala; Ile58Cys; Ile58Asp; Ile58Glu;Ile58Phe; Ile58Gly; Ile58His; Ile58Lys; Ile58Leu; Ile58Met; Ile58Asn;Ile58Pro; Ile58Gln; Ile58Arg; Ile58Ser; Ile58Thr; Ile58Val; Ile58Trp;Ile58Tyr; Glu59Ala; Glu59Cys; Glu59Asp; Glu59Phe; Glu59Gly; Glu59His;Glu59Ile; Glu59Lys; Glu59Leu; Glu59Met; Glu59Asn; Glu59Pro; Glu59Gln;Glu59Arg; Glu59Ser; Glu59Thr; Glu59Val; Glu59Trp; Glu59Tyr; Asp60Ala;Asp60Cys; Asp60Glu; Asp60Phe; Asp60Gly; Asp60His; Asp60Ile; Asp60Lys;Asp60Leu; Asp60Met; Asp60Asn; Asp60Pro; Asp60Gln; Asp60Arg; Asp60Ser;Asp60Thr; Asp60Val; Asp60Trp; Asp60Tyr; Leu61Ala; Leu61Cys; Leu61Asp;Leu61Glu; Leu61Phe; Leu61Gly; Leu61His; Leu61Ile; Leu61Lys; Leu61Met;Leu61Asn; Leu61Pro; Leu61Gln; Leu61Arg; Leu61Ser; Leu61Thr; Leu61Val;Leu61Trp; Leu61Tyr; Asn62Ala; Asn62Cys; Asn62Asp; Asn62Glu; Asn62Phe;Asn62Gly; Asn62His; Asn62Ile; Asn62Lys; Asn62Leu; Asn62Met; Asn62Pro;Asn62Gln; Asn62Arg; Asn62Ser; Asn62Thr; Asn62Val; Asn62Trp; Asn62Tyr;Gln63Ala; Gln63Cys; Gln63Asp; Gln63Glu; Gln63Phe; Gln63Gly; Gln63His;Gln63Ile; Gln63Lys; Gln63Leu; Gln63Met; Gln63Asn; Gln63Pro; Gln63Arg;Gln63Ser; Gln63Thr; Gln63Val; Gln63Trp; Gln63Tyr; Arg64Ala; Arg64Cys;Arg64Asp; Arg64Glu; Arg64Phe; Arg64Gly; Arg64His; Arg64Ile; Arg64Lys;Arg64Leu; Arg64Met; Arg64Asn; Arg64Pro; Arg64Gln; Arg64Ser; Arg64Thr;Arg64Val; Arg64Trp; Arg64Tyr; Ile65Ala; Ile65Cys; Ile65Asp; Ile65Glu;Ile65Phe; Ile65Gly; Ile65His; Ile65Lys; Ile65Leu; Ile65Met; Ile65Asn;Ile65Pro; Ile65Gln; Ile65Arg; Ile65Ser; Ile65Thr; Ile65Val; Ile65Trp;Ile65Tyr; Thr66Ala; Thr66Cys; Thr66Asp; Thr66Glu; Thr66Phe; Thr66Gly;Thr66His; Thr66Ile; Thr66Lys; Thr66Leu; Thr66Met; Thr66Asn; Thr66Pro;Thr66Gln; Thr66Arg; Thr66Ser; Thr66Val; Thr66Trp; Thr66Tyr; Tyr67Ala;Tyr67Cys; Tyr67Asp; Tyr67Glu; Tyr67Phe; Tyr67Gly; Tyr67His; Tyr67Ile;Tyr67Lys; Tyr67Leu; Tyr67Met; Tyr67Asn; Tyr67Pro; Tyr67Gln; Tyr67Arg;Tyr67Ser; Tyr67Thr; Tyr67Val; Tyr67Trp; Thr68Ala; Thr68Cys; Thr68Asp;Thr68Glu; Thr68Phe; Thr68Gly; Thr68His; Thr68Ile; Thr68Lys; Thr68Leu;Thr68Met; Thr68Asn; Thr68Pro; Thr68Gln; Thr68Arg; Thr68Ser; Thr68Val;Thr68Trp; Thr68Tyr; Arg69Ala; Arg69Cys; Arg69Asp; Arg69Glu; Arg69Phe;Arg69Gly; Arg69His; Arg69Ile; Arg69Lys; Arg69Leu; Arg69Met; Arg69Asn;Arg69Pro; Arg69Gln; Arg69Ser; Arg69Thr; Arg69Val; Arg69Trp; Arg69Tyr;Asp70Ala; Asp70Cys; Asp70Glu; Asp70Phe; Asp70Gly; Asp70His; Asp70Ile;Asp70Lys; Asp70Leu; Asp70Met; Asp70Asn; Asp70Pro; Asp70Gln; Asp70Arg;Asp70Ser; Asp70Thr; Asp70Val; Asp70Trp; Asp70Tyr; Asp71Ala; Asp71Cys;Asp71Glu; Asp71Phe; Asp71Gly; Asp71His; Asp71Ile; Asp71Lys; Asp71Leu;Asp71Met; Asp71Asn; Asp71Pro; Asp71Gln; Asp71Arg; Asp71Ser; Asp71Thr;Asp71Val; Asp71Trp; Asp71Tyr; Leu72Ala; Leu72Cys; Leu72Asp; Leu72Glu;Leu72Phe; Leu72Gly; Leu72His; Leu72Ile; Leu72Lys; Leu72Met; Leu72Asn;Leu72Pro; Leu72Gln; Leu72Arg; Leu72Ser; Leu72Thr; Leu72Val; Leu72Trp;Leu72Tyr; Val73Ala; Val73Cys; Val73Asp; Val73Glu; Val73Phe; Val73Gly;Val73His; Val73Ile; Val73Lys; Val73Leu; Val73Met; Val73Asn; Val73Pro;Val73Gln; Val73Arg; Val73Ser; Val73Thr; Val73Trp; Val73Tyr; Asn74Ala;Asn74Cys; Asn74Asp; Asn74Glu; Asn74Phe; Asn74Gly; Asn74His; Asn74Ile;Asn74Lys; Asn74Leu; Asn74Met; Asn74Pro; Asn74Gln; Asn74Arg; Asn74Ser;Asn74Thr; Asn74Val; Asn74Trp; Asn74Tyr; Tyr75Ala; Tyr75Cys; Tyr75Asp;Tyr75Glu; Tyr75Phe; Tyr75Gly; Tyr75His; Tyr75Ile; Tyr75Lys; Tyr75Leu;Tyr75Met; Tyr75Asn; Tyr75Pro; Tyr75Gln; Tyr75Arg; Tyr75Ser; Tyr75Thr;Tyr75Val; Tyr75Trp; Asn76Ala; Asn76Cys; Asn76Asp; Asn76Glu; Asn76Phe;Asn76Gly; Asn76His; Asn76Ile; Asn76Lys; Asn76Leu; Asn76Met; Asn76Pro;Asn76Gln; Asn76Arg; Asn76Ser; Asn76Thr; Asn76Val; Asn76Trp; Asn76Tyr;Pro77Ala; Pro77Cys; Pro77Asp; Pro77Glu; Pro77Phe; Pro77Gly; Pro77His;Pro77Ile; Pro77Lys; Pro77Leu; Pro77Met; Pro77Asn; Pro77Gln; Pro77Arg;Pro77Ser; Pro77Thr; Pro77Val; Pro77Trp; Pro77Tyr; Ile78Ala; Ile78Cys;Ile78Asp; Ile78Glu; Ile78Phe; Ile78Gly; Ile78His; Ile78Lys; Ile78Leu;Ile78Met; Ile78Asn; Ile78Pro; Ile78Gln; Ile78Arg; Ile78Ser; Ile78Thr;Ile78Val; Ile78Trp; Ile78Tyr; Thr79Ala; Thr79Cys; Thr79Asp; Thr79Glu;Thr79Phe; Thr79Gly; Thr79His; Thr79Ile; Thr79Lys; Thr79Leu; Thr79Met;Thr79Asn; Thr79Pro; Thr79Gln; Thr79Arg; Thr79Ser; Thr79Val; Thr79Trp;Thr79Tyr; Glu80Ala; Glu80Cys; Glu80Asp; Glu80Phe; Glu80Gly; Glu80His;Glu80Ile; Glu80Lys; Glu80Leu; Glu80Met; Glu80Asn; Glu80Pro; Glu80Gln;Glu80Arg; Glu80Ser; Glu80Thr; Glu80Val; Glu80Trp; Glu80Tyr; Lys81Ala;Lys81Cys; Lys81Asp; Lys81Glu; Lys81Phe; Lys81Gly; Lys81His; Lys81Ile;Lys81Leu; Lys81Met; Lys81Asn; Lys81Pro; Lys81Gln; Lys81Arg; Lys81Ser;Lys81Thr; Lys81Val; Lys81Trp; Lys81Tyr; His82Ala; His82Cys; His82Asp;His82Glu; His82Phe; His82Gly; His82Ile; His82Lys; His82Leu; His82Met;His82Asn; His82Pro; His82Gln; His82Arg; His82Ser; His82Thr; His82Val;His82Trp; His82Tyr; Val83Ala; Val83Cys; Val83Asp; Val83Glu; Val83Phe;Val83Gly; Val83His; Val83Ile; Val83Lys; Val83Leu; Val83Met; Val83Asn;Val83Pro; Val83Gln; Val83Arg; Val83Ser; Val83Thr; Val83Trp; Val83Tyr;Asp84Ala; Asp84Cys; Asp84Glu; Asp84Phe; Asp84Gly; Asp84His; Asp84Ile;Asp84Lys; Asp84Leu; Asp84Met; Asp84Asn; Asp84Pro; Asp84Gln; Asp84Arg;Asp84Ser; Asp84Thr; Asp84Val; Asp84Trp; Asp84Tyr; Thr85Ala; Thr85Cys;Thr85Asp; Thr85Glu; Thr85Phe; Thr85Gly; Thr85His; Thr85Ile; Thr85Lys;Thr85Leu; Thr85Met; Thr85Asn; Thr85Pro; Thr85Gln; Thr85Arg; Thr85Ser;Thr85Val; Thr85Trp; Thr85Tyr; Gly86Ala; Gly86Cys; Gly86Asp; Gly86Glu;Gly86Phe; Gly86His; Gly86Ile; Gly86Lys; Gly86Leu; Gly86Met; Gly86Asn;Gly86Pro; Gly86Gln; Gly86Arg; Gly86Ser; Gly86Thr; Gly86Val; Gly86Trp;Gly86Tyr; Met87Ala; Met87Cys; Met87Asp; Met87Glu; Met87Phe; Met87Gly;Met87His; Met87Ile; Met87Lys; Met87Leu; Met87Asn; Met87Pro; Met87Gln;Met87Arg; Met87Ser; Met87Thr; Met87Val; Met87Trp; Met87Tyr; Thr88Ala;Thr88Cys; Thr88Asp; Thr88Glu; Thr88Phe; Thr88Gly; Thr88His; Thr88Ile;Thr88Lys; Thr88Leu; Thr88Met; Thr88Asn; Thr88Pro; Thr88Gln; Thr88Arg;Thr88Ser; Thr88Val; Thr88Trp; Thr88Tyr; Leu89Ala; Leu89Cys; Leu89Asp;Leu89Glu; Leu89Phe; Leu89Gly; Leu89His; Leu89Ile; Leu89Lys; Leu89Met;Leu89Asn; Leu89Pro; Leu89Gln; Leu89Arg; Leu89Ser; Leu89Thr; Leu89Val;Leu89Trp; Leu89Tyr; Lys90Ala; Lys90Cys; Lys90Asp; Lys90Glu; Lys90Phe;Lys90Gly; Lys90His; Lys90Ile; Lys90Leu; Lys90Met; Lys90Asn; Lys90Pro;Lys90Gln; Lys90Arg; Lys90Ser; Lys90Thr; Lys90Val; Lys90Trp; Lys90Tyr;Glu91Ala; Glu91Cys; Glu91Asp; Glu91Phe; Glu91Gly; Glu91His; Glu91Ile;Glu91Lys; Glu91Leu; Glu91Met; Glu91Asn; Glu91Pro; Glu91Gln; Glu91Arg;Glu91Ser; Glu91Thr; Glu91Val; Glu91Trp; Glu91Tyr; Leu92Ala; Leu92Cys;Leu92Asp; Leu92Glu; Leu92Phe; Leu92Gly; Leu92His; Leu92Ile; Leu92Lys;Leu92Met; Leu92Asn; Leu92Pro; Leu92Gln; Leu92Arg; Leu92Ser; Leu92Thr;Leu92Val; Leu92Trp; Leu92Tyr; Ala93Cys; Ala93Asp; Ala93Glu; Ala93Phe;Ala93Gly; Ala93His; Ala93Ile; Ala93Lys; Ala93Leu; Ala93Met; Ala93Asn;Ala93Pro; Ala93Gln; Ala93Arg; Ala93Ser; Ala93Thr; Ala93Val; Ala93Trp;Ala93Tyr; Asp94Ala; Asp94Cys; Asp94Glu; Asp94Phe; Asp94Gly; Asp94His;Asp94Ile; Asp94Lys; Asp94Leu; Asp94Met; Asp94Asn; Asp94Pro; Asp94Gln;Asp94Arg; Asp94Ser; Asp94Thr; Asp94Val; Asp94Trp; Asp94Tyr; Ala95Cys;Ala95Asp; Ala95Glu; Ala95Phe; Ala95Gly; Ala95His; Ala95Ile; Ala95Lys;Ala95Leu; Ala95Met; Ala95Asn; Ala95Pro; Ala95Gln; Ala95Arg; Ala95Ser;Ala95Thr; Ala95Val; Ala95Trp; Ala95Tyr; Ser96Ala; Ser96Cys; Ser96Asp;Ser96Glu; Ser96Phe; Ser96Gly; Ser96His; Ser96Ile; Ser96Lys; Ser96Leu;Ser96Met; Ser96Asn; Ser96Pro; Ser96Gln; Ser96Arg; Ser96Thr; Ser96Val;Ser96Trp; Ser96Tyr; Leu97Ala; Leu97Cys; Leu97Asp; Leu97Glu; Leu97Phe;Leu97Gly; Leu97His; Leu97Ile; Leu97Lys; Leu97Met; Leu97Asn; Leu97Pro;Leu97Gln; Leu97Arg; Leu97Ser; Leu97Thr; Leu97Val; Leu97Trp; Leu97Tyr;Arg98Ala; Arg98Cys; Arg98Asp; Arg98Glu; Arg98Phe; Arg98Gly; Arg98His;Arg98Ile; Arg98Lys; Arg98Leu; Arg98Met; Arg98Asn; Arg98Pro; Arg98Gln;Arg98Ser; Arg98Thr; Arg98Val; Arg98Trp; Arg98Tyr; Tyr99Ala; Tyr99Cys;Tyr99Asp; Tyr99Glu; Tyr99Phe; Tyr99Gly; Tyr99His; Tyr99Ile; Tyr99Lys;Tyr99Leu; Tyr99Met; Tyr99Asn; Tyr99Pro; Tyr99Gln; Tyr99Arg; Tyr99Ser;Tyr99Thr; Tyr99Val; Tyr99Trp; Ser100Ala; Ser100Cys; Ser100Asp;Ser100Glu; Ser100Phe; Ser100Gly; Ser100His; Ser100Ile; Ser100Lys;Ser100Leu; Ser100Met; Ser100Asn; Ser100Pro; Ser100Gln; Ser100Arg;Ser100Thr; Ser100Val; Ser100Trp; Ser100Tyr; Asp101Ala; Asp101Cys;Asp101Glu; Asp101Phe; Asp101Gly; Asp101His; Asp101Ile; Asp101Lys;Asp101Leu; Asp101Met; Asp101Asn; Asp101Pro; Asp101Gln; Asp101Arg;Asp101Ser; Asp101Thr; Asp101Val; Asp101Trp; Asp101Tyr; Asn102Ala;Asn102Cys; Asn102Asp; Asn102Glu; Asn102Phe; Asn102Gly; Asn102His;Asn102Ile; Asn102Lys; Asn102Leu; Asn102Met; Asn102Pro; Asn102Gln;Asn102Arg; Asn102Ser; Asn102Thr; Asn102Val; Asn102Trp; Asn102Tyr;Ala103Cys; Ala103Asp; Ala103Glu; Ala103Phe; Ala103Gly; Ala103His;Ala103Ile; Ala103Lys; Ala103Leu; Ala103Met; Ala103Asn; Ala103Pro;Ala103Gln; Ala103Arg; Ala103Ser; Ala103Thr; Ala103Val; Ala103Trp;Ala103Tyr; Ala104Cys; Ala104Asp; Ala104Glu; Ala104Phe; Ala104Gly;Ala104His; Ala104Ile; Ala104Lys; Ala104Leu; Ala104Met; Ala104Asn;Ala104Pro; Ala104Gln; Ala104Arg; Ala104Ser; Ala104Thr; Ala104Val;Ala104Trp; Ala104Tyr; Gln105Ala; Gln105Cys; Gln105Asp; Gln105Glu;Gln105Phe; Gln105Gly; Gln105His; Gln105Ile; Gln105Lys; Gln105Leu;Gln105Met; Gln105Asn; Gln105Pro; Gln105Arg; Gln105Ser; Gln105Thr;Gln105Val; Gln105Trp; Gln105Tyr; Asn106Ala; Asn106Cys; Asn106Asp;Asn106Glu; Asn106Phe; Asn106Gly; Asn106His; Asn106Ile; Asn106Lys;Asn106Leu; Asn106Met; Asn106Pro; Asn106Gln; Asn106Arg; Asn106Ser;Asn106Thr; Asn106Val; Asn106Trp; Asn106Tyr; Leu107Ala; Leu107Cys;Leu107Asp; Leu107Glu; Leu107Phe; Leu107Gly; Leu107His; Leu107Ile;Leu107Lys; Leu107Met; Leu107Asn; Leu107Pro; Leu107Gln; Leu107Arg;Leu107Ser; Leu107Thr; Leu107Val; Leu107Trp; Leu107Tyr; Ile108Ala;Ile108Cys; Ile108Asp; Ile108Glu; Ile108Phe; Ile108Gly; Ile108His;Ile108Lys; Ile108Leu; Ile108Met; Ile108Asn; Ile108Pro; Ile108Gln;Ile108Arg; Ile108Ser; Ile108Thr; Ile108Val; Ile108Trp; Ile108Tyr;Leu109Ala; Leu109Cys; Leu109Asp; Leu109Glu; Leu109Phe; Leu109Gly;Leu109His; Leu109Ile; Leu109Lys; Leu109Met; Leu109Asn; Leu109Pro;Leu109Gln; Leu109Arg; Leu109Ser; Leu109Thr; Leu109Val; Leu109Trp;Leu109Tyr; Lys110Ala; Lys110Cys; Lys110Asp; Lys110Glu; Lys110Phe;Lys110Gly; Lys110His; Lys110Ile; Lys110Leu; Lys110Met; Lys110Asn;Lys110Pro; Lys110Gln; Lys110Arg; Lys110Ser; Lys110Thr; Lys110Val;Lys110Trp; Lys110Tyr; Gln111Ala; Gln111Cys; Gln111Asp; Gln111Glu;Gln111Phe; Gln111Gly; Gln111His; Gln111Ile; Gln111Lys; Gln111Leu;Gln111Met; Gln111Asn; Gln111Pro; Gln111Arg; Gln111Ser; Gln111Thr;Gln111Val; Gln111Trp; Gln111Tyr; Ile112Ala; Ile112Cys; Ile112Asp;Ile112Glu; Ile112Phe; Ile112Gly; Ile112His; Ile112Lys; Ile112Leu;Ile112Met; Ile112Asn; Ile112Pro; Ile112Gln; Ile112Arg; Ile112Ser;Ile112Thr; Ile112Val; Ile112Trp; Ile112Tyr; Gly113Ala; Gly113Cys;Gly113Asp; Gly113Glu; Gly113Phe; Gly113His; Gly113Ile; Gly113Lys;Gly113Leu; Gly113Met; Gly113Asn; Gly113Pro; Gly113Gln; Gly113Arg;Gly113Ser; Gly113Thr; Gly113Val; Gly113Trp; Gly113Tyr; Gly114Ala;Gly114Cys; Gly114Asp; Gly114Glu; Gly114Phe; Gly114His; Gly114Ile;Gly114Lys; Gly114Leu; Gly114Met; Gly114Asn; Gly114Pro; Gly114Gln;Gly114Arg; Gly114Ser; Gly114Thr; Gly114Val; Gly114Trp; Gly114Tyr;Pro115Ala; Pro115Cys; Pro115Asp; Pro115Glu; Pro115Phe; Pro115Gly;Pro115His; Pro115Ile; Pro115Lys; Pro115Leu; Pro115Met; Pro115Asn;Pro115Gln; Pro115Arg; Pro115Ser; Pro115Thr; Pro115Val; Pro115Trp;Pro115Tyr; Glu116Ala; Glu116Cys; Glu116Asp; Glu116Phe; Glu116Gly;Glu116His; Glu116Ile; Glu116Lys; Glu116Leu; Glu116Met; Glu116Asn;Glu116Pro; Glu116Gln; Glu116Arg; Glu116Ser; Glu116Thr; Glu116Val;Glu116Trp; Glu116Tyr; Ser117Ala; Ser117Cys; Ser117Asp; Ser117Glu;Ser117Phe; Ser117Gly; Ser117His; Ser117Ile; Ser117Lys; Ser117Leu;Ser117Met; Ser117Asn; Ser117Pro; Ser117Gln; Ser117Arg; Ser117Thr;Ser117Val; Ser117Trp; Ser117Tyr; Leu118Ala; Leu118Cys; Leu118Asp;Leu118Glu; Leu118Phe; Leu118Gly; Leu118His; Leu118Ile; Leu118Lys;Leu118Met; Leu118Asn; Leu118Pro; Leu118Gln; Leu118Arg; Leu118Ser;Leu118Thr; Leu118Val; Leu118Trp; Leu118Tyr; Lys119Ala; Lys119Cys;Lys119Asp; Lys119Glu; Lys119Phe; Lys119Gly; Lys119His; Lys119Ile;Lys119Leu; Lys119Met; Lys119Asn; Lys119Pro; Lys119Gln; Lys119Arg;Lys119Ser; Lys119Thr; Lys119Val; Lys119Trp; Lys119Tyr; Lys120Ala;Lys120Cys; Lys120Asp; Lys120Glu; Lys120Phe; Lys120Gly; Lys120His;Lys120Ile; Lys120Leu; Lys120Met; Lys120Asn; Lys120Pro; Lys120Gln;Lys120Arg; Lys120Ser; Lys120Thr; Lys120Val; Lys120Trp; Lys120Tyr;Glu121Ala; Glu121Cys; Glu121Asp; Glu121Phe; Glu121Gly; Glu121His;Glu121Ile; Glu121Lys; Glu121Leu; Glu121Met; Glu121Asn; Glu121Pro;Glu121Gln; Glu121Arg; Glu121Ser; Glu121Thr; Glu121Val; Glu121Trp;Glu121Tyr; Leu122Ala; Leu122Cys; Leu122Asp; Leu122Glu; Leu122Phe;Leu122Gly; Leu122His; Leu122Ile; Leu122Lys; Leu122Met; Leu122Asn;Leu122Pro; Leu122Gln; Leu122Arg; Leu122Ser; Leu122Thr; Leu122Val;Leu122Trp; Leu122Tyr; Arg123Ala; Arg123Cys; Arg123Asp; Arg123Glu;Arg123Phe; Arg123Gly; Arg123His; Arg123Ile; Arg123Lys; Arg123Leu;Arg123Met; Arg123Asn; Arg123Pro; Arg123Gln; Arg123Ser; Arg123Thr;Arg123Val; Arg123Trp; Arg123Tyr; Lys124Ala; Lys124Cys; Lys124Asp;Lys124Glu; Lys124Phe; Lys124Gly; Lys124His; Lys124Ile; Lys124Leu;Lys124Met; Lys124Asn; Lys124Pro; Lys124Gln; Lys124Arg; Lys124Ser;Lys124Thr; Lys124Val; Lys124Trp; Lys124Tyr; Ile125Ala; Ile125Cys;Ile125Asp; Ile125Glu; Ile125Phe; Ile125Gly; Ile125His; Ile125Lys;Ile125Leu; Ile125Met; Ile125Asn; Ile125Pro; Ile125Gln; Ile125Arg;Ile125Ser; Ile125Thr; Ile125Val; Ile125Trp; Ile125Tyr; Gly126Ala;Gly126Cys; Gly126Asp; Gly126Glu; Gly126Phe; Gly126His; Gly126Ile;Gly126Lys; Gly126Leu; Gly126Met; Gly126Asn; Gly126Pro; Gly126Gln;Gly126Arg; Gly126Ser; Gly126Thr; Gly126Val; Gly126Trp; Gly126Tyr;Asp127Ala; Asp127Cys; Asp127Glu; Asp127Phe; Asp127Gly; Asp127His;Asp127Ile; Asp127Lys; Asp127Leu; Asp127Met; Asp127Asn; Asp127Pro;Asp127Gln; Asp127Arg; Asp127Ser; Asp127Thr; Asp127Val; Asp127Trp;Asp127Tyr; Glu128Ala; Glu128Cys; Glu128Asp; Glu128Phe; Glu128Gly;Glu128His; Glu128Ile; Glu128Lys; Glu128Leu; Glu128Met; Glu128Asn;Glu128Pro; Glu128Gln; Glu128Arg; Glu128Ser; Glu128Thr; Glu128Val;Glu128Trp; Glu128Tyr; Val129Ala; Val129Cys; Val129Asp; Val129Glu;Val129Phe; Val129Gly; Val129His; Val129Ile; Val129Lys; Val129Leu;Val129Met; Val129Asn; Val129Pro; Val129Gln; Val129Arg; Val129Ser;Val129Thr; Val129Trp; Val129Tyr; Thr130Ala; Thr130Cys; Thr130Asp;Thr130Glu; Thr130Phe; Thr130Gly; Thr130His; Thr130Ile; Thr130Lys;Thr130Leu; Thr130Met; Thr130Asn; Thr130Pro; Thr130Gln; Thr130Arg;Thr130Ser; Thr130Val; Thr130Trp; Thr130Tyr; Asn131Ala; Asn131Cys;Asn131Asp; Asn131Glu; Asn131Phe; Asn131Gly; Asn131His; Asn131Ile;Asn131Lys; Asn131Leu; Asn131Met; Asn131Pro; Asn131Gln; Asn131Arg;Asn131Ser; Asn131Thr; Asn131Val; Asn131Trp; Asn131Tyr; Pro132Ala;Pro132Cys; Pro132Asp; Pro132Glu; Pro132Phe; Pro132Gly; Pro132His;Pro132Ile; Pro132Lys; Pro132Leu; Pro132Met; Pro132Asn; Pro132Gln;Pro132Arg; Pro132Ser; Pro132Thr; Pro132Val; Pro132Trp; Pro132Tyr;Glu133Ala; Glu133Cys; Glu133Asp; Glu133Phe; Glu133Gly; Glu133His;Glu133Ile; Glu133Lys; Glu133Leu; Glu133Met; Glu133Asn; Glu133Pro;Glu133Gln; Glu133Arg; Glu133Ser; Glu133Thr; Glu133Val; Glu133Trp;Glu133Tyr; Arg134Ala; Arg134Cys; Arg134Asp; Arg134Glu; Arg134Phe;Arg134Gly; Arg134His; Arg134Ile; Arg134Lys; Arg134Leu; Arg134Met;Arg134Asn; Arg134Pro; Arg134Gln; Arg134Ser; Arg134Thr; Arg134Val;Arg134Trp; Arg134Tyr; Phe135Ala; Phe135Cys; Phe135Asp; Phe135Glu;Phe135Gly; Phe135His; Phe135Ile; Phe135Lys; Phe135Leu; Phe135Met;Phe135Asn; Phe135Pro; Phe135Gln; Phe135Arg; Phe135Ser; Phe135Thr;Phe135Val; Phe135Trp; Phe135Tyr; Glu136Ala; Glu136Cys; Glu136Asp;Glu136Phe; Glu136Gly; Glu136His; Glu136Ile; Glu136Lys; Glu136Leu;Glu136Met; Glu136Asn; Glu136Pro; Glu136Gln; Glu136Arg; Glu136Ser;Glu136Thr; Glu136Val; Glu136Trp; Glu136Tyr; Pro137Ala; Pro137Cys;Pro137Asp; Pro137Glu; Pro137Phe; Pro137Gly; Pro137His; Pro137Ile;Pro137Lys; Pro137Leu; Pro137Met; Pro137Asn; Pro137Gln; Pro137Arg;Pro137Ser; Pro137Thr; Pro137Val; Pro137Trp; Pro137Tyr; Glu138Ala;Glu138Cys; Glu138Asp; Glu138Phe; Glu138Gly; Glu138His; Glu138Ile;Glu138Lys; Glu138Leu; Glu138Met; Glu138Asn; Glu138Pro; Glu138Gln;Glu138Arg; Glu138Ser; Glu138Thr; Glu138Val; Glu138Trp; Glu138Tyr;Leu139Ala; Leu139Cys; Leu139Asp; Leu139Glu; Leu139Phe; Leu139Gly;Leu139His; Leu139Ile; Leu139Lys; Leu139Met; Leu139Asn; Leu139Pro;Leu139Gln; Leu139Arg; Leu139Ser; Leu139Thr; Leu139Val; Leu139Trp;Leu139Tyr; Asn140Ala; Asn140Cys; Asn140Asp; Asn140Glu; Asn140Phe;Asn140Gly; Asn140His; Asn140Ile; Asn140Lys; Asn140Leu; Asn140Met;Asn140Pro; Asn140Gln; Asn140Arg; Asn140Ser; Asn140Thr; Asn140Val;Asn140Trp; Asn140Tyr; Glu141Ala; Glu141Cys; Glu141Asp; Glu141Phe;Glu141Gly; Glu141His; Glu141Ile; Glu141Lys; Glu141Leu; Glu141Met;Glu141Asn; Glu141Pro; Glu141Gln; Glu141Arg; Glu141Ser; Glu141Thr;Glu141Val; Glu141Trp; Glu141Tyr; Val142Ala; Val142Cys; Val142Asp;Val142Glu; Val142Phe; Val142Gly; Val142His; Val142Ile; Val142Lys;Val142Leu; Val142Met; Val142Asn; Val142Pro; Val142Gln; Val142Arg;Val142Ser; Val142Thr; Val142Trp; Val142Tyr; Asn143Ala; Asn143Cys;Asn143Asp; Asn143Glu; Asn143Phe; Asn143Gly; Asn143His; Asn143Ile;Asn143Lys; Asn143Leu; Asn143Met; Asn143Pro; Asn143Gln; Asn143Arg;Asn143Ser; Asn143Thr; Asn143Val; Asn143Trp; Asn143Tyr; Pro144Ala;Pro144Cys; Pro144Asp; Pro144Glu; Pro144Phe; Pro144Gly; Pro144His;Pro144Ile; Pro144Lys; Pro144Leu; Pro144Met; Pro144Asn; Pro144Gln;Pro144Arg; Pro144Ser; Pro144Thr; Pro144Val; Pro144Trp; Pro144Tyr;Gly145Ala; Gly145Cys; Gly145Asp; Gly145Glu; Gly145Phe; Gly145His;Gly145Ile; Gly145Lys; Gly145Leu; Gly145Met; Gly145Asn; Gly145Pro;Gly145Gln; Gly145Arg; Gly145Ser; Gly145Thr; Gly145Val; Gly145Trp;Gly145Tyr; Glu146Ala; Glu146Cys; Glu146Asp; Glu146Phe; Glu146Gly;Glu146His; Glu146Ile; Glu146Lys; Glu146Leu; Glu146Met; Glu146Asn;Glu146Pro; Glu146Gln; Glu146Arg; Glu146Ser; Glu146Thr; Glu146Val;Glu146Trp; Glu146Tyr; Thr147Ala; Thr147Cys; Thr147Asp; Thr147Glu;Thr147Phe; Thr147Gly; Thr147His; Thr147Ile; Thr147Lys; Thr147Leu;Thr147Met; Thr147Asn; Thr147Pro; Thr147Gln; Thr147Arg; Thr147Ser;Thr147Val; Thr147Trp; Thr147Tyr; Gln148Ala; Gln148Cys; Gln148Asp;Gln148Glu; Gln148Phe; Gln148Gly; Gln148His; Gln148Ile; Gln148Lys;Gln148Leu; Gln148Met; Gln148Asn; Gln148Pro; Gln148Arg; Gln148Ser;Gln148Thr; Gln148Val; Gln148Trp; Gln148Tyr; Asp149Ala; Asp149Cys;Asp149Glu; Asp149Phe; Asp149Gly; Asp149His; Asp149Ile; Asp149Lys;Asp149Leu; Asp149Met; Asp149Asn; Asp149Pro; Asp149Gln; Asp149Arg;Asp149Ser; Asp149Thr; Asp149Val; Asp149Trp; Asp149Tyr; Thr150Ala;Thr150Cys; Thr150Asp; Thr150Glu; Thr150Phe; Thr150Gly; Thr150His;Thr150Ile; Thr150Lys; Thr150Leu; Thr150Met; Thr150Asn; Thr150Pro;Thr150Gln; Thr150Arg; Thr150Ser; Thr150Val; Thr150Trp; Thr150Tyr;Ser151Ala; Ser151Cys; Ser151Asp; Ser151Glu; Ser151Phe; Ser151Gly;Ser151His; Ser151Ile; Ser151Lys; Ser151Leu; Ser151Met; Ser151Asn;Ser151Pro; Ser151Gln; Ser151Arg; Ser151Thr; Ser151Val; Ser151Trp;Ser151Tyr; Thr152Ala; Thr152Cys; Thr152Asp; Thr152Glu; Thr152Phe;Thr152Gly; Thr152His; Thr152Ile; Thr152Lys; Thr152Leu; Thr152Met;Thr152Asn; Thr152Pro; Thr152Gln; Thr152Arg; Thr152Ser; Thr152Val;Thr152Trp; Thr152Tyr; Ala153Cys; Ala153Asp; Ala153Glu; Ala153Phe;Ala153Gly; Ala153His; Ala153Ile; Ala153Lys; Ala153Leu; Ala153Met;Ala153Asn; Ala153Pro; Ala153Gln; Ala153Arg; Ala153Ser; Ala153Thr;Ala153Val; Ala153Trp; Ala153Tyr; Arg154Ala; Arg154Cys; Arg154Asp;Arg154Glu; Arg154Phe; Arg154Gly; Arg154His; Arg154Ile; Arg154Lys;Arg154Leu; Arg154Met; Arg154Asn; Arg154Pro; Arg154Gln; Arg154Ser;Arg154Thr; Arg154Val; Arg154Trp; Arg154Tyr; Ala155Cys; Ala155Asp;Ala155Glu; Ala155Phe; Ala155Gly; Ala155His; Ala155Ile; Ala155Lys;Ala155Leu; Ala155Met; Ala155Asn; Ala155Pro; Ala155Gln; Ala155Arg;Ala155Ser; Ala155Thr; Ala155Val; Ala155Trp; Ala155Tyr; Leu156Ala;Leu156Cys; Leu156Asp; Leu156Glu; Leu156Phe; Leu156Gly; Leu156His;Leu156Ile; Leu156Lys; Leu156Met; Leu156Asn; Leu156Pro; Leu156Gln;Leu156Arg; Leu156Ser; Leu156Thr; Leu156Val; Leu156Trp; Leu156Tyr;Val157Ala; Val157Cys; Val157Asp; Val157Glu; Val157Phe; Val157Gly;Val157His; Val157Ile; Val157Lys; Val157Leu; Val157Met; Val157Asn;Val157Pro; Val157Gln; Val157Arg; Val157Ser; Val157Thr; Val157Trp;Val157Tyr; Thr158Ala; Thr158Cys; Thr158Asp; Thr158Glu; Thr158Phe;Thr158Gly; Thr158His; Thr158Ile; Thr158Lys; Thr158Leu; Thr158Met;Thr158Asn; Thr158Pro; Thr158Gln; Thr158Arg; Thr158Ser; Thr158Val;Thr158Trp; Thr158Tyr; Ser159Ala; Ser159Cys; Ser159Asp; Ser159Glu;Ser159Phe; Ser159Gly; Ser159His; Ser159Ile; Ser159Lys; Ser159Leu;Ser159Met; Ser159Asn; Ser159Pro; Ser159Gln; Ser159Arg; Ser159Thr;Ser159Val; Ser159Trp; Ser159Tyr; Leu160Ala; Leu160Cys; Leu160Asp;Leu160Glu; Leu160Phe; Leu160Gly; Leu160His; Leu160Ile; Leu160Lys;Leu160Met; Leu160Asn; Leu160Pro; Leu160Gln; Leu160Arg; Leu160Ser;Leu160Thr; Leu160Val; Leu160Trp; Leu160Tyr; Arg161Ala; Arg161Cys;Arg161Asp; Arg161Glu; Arg161Phe; Arg161Gly; Arg161His; Arg161Ile;Arg161Lys; Arg161Leu; Arg161Met; Arg161Asn; Arg161Pro; Arg161Gln;Arg161Ser; Arg161Thr; Arg161Val; Arg161Trp; Arg161Tyr; Ala162Cys;Ala162Asp; Ala162Glu; Ala162Phe; Ala162Gly; Ala162His; Ala162Ile;Ala162Lys; Ala162Leu; Ala162Met; Ala162Asn; Ala162Pro; Ala162Gln;Ala162Arg; Ala162Ser; Ala162Thr; Ala162Val; Ala162Trp; Ala162Tyr;Phe163Ala; Phe163Cys; Phe163Asp; Phe163Glu; Phe163Gly; Phe163His;Phe163Ile; Phe163Lys; Phe163Leu; Phe163Met; Phe163Asn; Phe163Pro;Phe163Gln; Phe163Arg; Phe163Ser; Phe163Thr; Phe163Val; Phe163Trp;Phe163Tyr; Ala164Cys; Ala164Asp; Ala164Glu; Ala164Phe; Ala164Gly;Ala164His; Ala164Ile; Ala164Lys; Ala164Leu; Ala164Met; Ala164Asn;Ala164Pro; Ala164Gln; Ala164Arg; Ala164Ser; Ala164Thr; Ala164Val;Ala164Trp; Ala164Tyr; Leu165Ala; Leu165Cys; Leu165Asp; Leu165Glu;Leu165Phe; Leu165Gly; Leu165His; Leu165Ile; Leu165Lys; Leu165Met;Leu165Asn; Leu165Pro; Leu165Gln; Leu165Arg; Leu165Ser; Leu165Thr;Leu165Val; Leu165Trp; Leu165Tyr; Glu166Ala; Glu166Cys; Glu166Asp;Glu166Phe; Glu166Gly; Glu166His; Glu166Ile; Glu166Lys; Glu166Leu;Glu166Met; Glu166Asn; Glu166Pro; Glu166Gln; Glu166Arg; Glu166Ser;Glu166Thr; Glu166Val; Glu166Trp; Glu166Tyr; Asp167Ala; Asp167Cys;Asp167Glu; Asp167Phe; Asp167Gly; Asp167His; Asp167Ile; Asp167Lys;Asp167Leu; Asp167Met; Asp167Asn; Asp167Pro; Asp167Gln; Asp167Arg;Asp167Ser; Asp167Thr; Asp167Val; Asp167Trp; Asp167Tyr; Lys168Ala;Lys168Cys; Lys168Asp; Lys168Glu; Lys168Phe; Lys168Gly; Lys168His;Lys168Ile; Lys168Leu; Lys168Met; Lys168Asn; Lys168Pro; Lys168Gln;Lys168Arg; Lys168Ser; Lys168Thr; Lys168Val; Lys168Trp; Lys168Tyr;Leu169Ala; Leu169Cys; Leu169Asp; Leu169Glu; Leu169Phe; Leu169Gly;Leu169His; Leu169Ile; Leu169Lys; Leu169Met; Leu169Asn; Leu169Pro;Leu169Gln; Leu169Arg; Leu169Ser; Leu169Thr; Leu169Val; Leu169Trp;Leu169Tyr; Pro170Ala; Pro170Cys; Pro170Asp; Pro170Glu; Pro170Phe;Pro170Gly; Pro170His; Pro170Ile; Pro170Lys; Pro170Leu; Pro170Met;Pro170Asn; Pro170Gln; Pro170Arg; Pro170Ser; Pro170Thr; Pro170Val;Pro170Trp; Pro170Tyr; Ser171Ala; Ser171Cys; Ser171Asp; Ser171Glu;Ser171Phe; Ser171Gly; Ser171His; Ser171Ile; Ser171Lys; Ser171Leu;Ser171Met; Ser171Asn; Ser171Pro; Ser171Gln; Ser171Arg; Ser171Thr;Ser171Val; Ser171Trp; Ser171Tyr; Glu172Ala; Glu172Cys; Glu172Asp;Glu172Phe; Glu172Gly; Glu172His; Glu172Ile; Glu172Lys; Glu172Leu;Glu172Met; Glu172Asn; Glu172Pro; Glu172Gln; Glu172Arg; Glu172Ser;Glu172Thr; Glu172Val; Glu172Trp; Glu172Tyr; Lys173Ala; Lys173Cys;Lys173Asp; Lys173Glu; Lys173Phe; Lys173Gly; Lys173His; Lys173Ile;Lys173Leu; Lys173Met; Lys173Asn; Lys173Pro; Lys173Gln; Lys173Arg;Lys173Ser; Lys173Thr; Lys173Val; Lys173Trp; Lys173Tyr; Arg174Ala;Arg174Cys; Arg174Asp; Arg174Glu; Arg174Phe; Arg174Gly; Arg174His;Arg174Ile; Arg174Lys; Arg174Leu; Arg174Met; Arg174Asn; Arg174Pro;Arg174Gln; Arg174Ser; Arg174Thr; Arg174Val; Arg174Trp; Arg174Tyr;Glu175Ala; Glu175Cys; Glu175Asp; Glu175Phe; Glu175Gly; Glu175His;Glu175Ile; Glu175Lys; Glu175Leu; Glu175Met; Glu175Asn; Glu175Pro;Glu175Gln; Glu175Arg; Glu175Ser; Glu175Thr; Glu175Val; Glu175Trp;Glu175Tyr; Leu176Ala; Leu176Cys; Leu176Asp; Leu176Glu; Leu176Phe;Leu176Gly; Leu176His; Leu176Ile; Leu176Lys; Leu176Met; Leu176Asn;Leu176Pro; Leu176Gln; Leu176Arg; Leu176Ser; Leu176Thr; Leu176Val;Leu176Trp; Leu176Tyr; Leu177Ala; Leu177Cys; Leu177Asp; Leu177Glu;Leu177Phe; Leu177Gly; Leu177His; Leu177Ile; Leu177Lys; Leu177Met;Leu177Asn; Leu177Pro; Leu177Gln; Leu177Arg; Leu177Ser; Leu177Thr;Leu177Val; Leu177Trp; Leu177Tyr; Ile178Ala; Ile178Cys; Ile178Asp;Ile178Glu; Ile178Phe; Ile178Gly; Ile178His; Ile178Lys; Ile178Leu;Ile178Met; Ile178Asn; Ile178Pro; Ile178Gln; Ile178Arg; Ile178Ser;Ile178Thr; Ile178Val; Ile178Trp; Ile178Tyr; Asp179Ala; Asp179Cys;Asp179Glu; Asp179Phe; Asp179Gly; Asp179His; Asp179Ile; Asp179Lys;Asp179Leu; Asp179Met; Asp179Asn; Asp179Pro; Asp179Gln; Asp179Arg;Asp179Ser; Asp179Thr; Asp179Val; Asp179Trp; Asp179Tyr; Trp180Ala;Trp180Cys; Trp180Asp; Trp180Glu; Trp180Phe; Trp180Gly; Trp180His;Trp180Ile; Trp180Lys; Trp180Leu; Trp180Met; Trp180Asn; Trp180Pro;Trp180Gln; Trp180Arg; Trp180Ser; Trp180Thr; Trp180Val; Trp180Tyr;Met181Ala; Met181Cys; Met181Asp; Met181Glu; Met181Phe; Met181Gly;Met181His; Met181Ile; Met181Lys; Met181Leu; Met181Asn; Met181Pro;Met181Gln; Met181Arg; Met181Ser; Met181Thr; Met181Val; Met181Trp;Met181Tyr; Lys182Ala; Lys182Cys; Lys182Asp; Lys182Glu; Lys182Phe;Lys182Gly; Lys182His; Lys182Ile; Lys182Leu; Lys182Met; Lys182Asn;Lys182Pro; Lys182Gln; Lys182Arg; Lys182Ser; Lys182Thr; Lys182Val;Lys182Trp; Lys182Tyr; Arg183Ala; Arg183Cys; Arg183Asp; Arg183Glu;Arg183Phe; Arg183Gly; Arg183His; Arg183Ile; Arg183Lys; Arg183Leu;Arg183Met; Arg183Asn; Arg183Pro; Arg183Gln; Arg183Ser; Arg183Thr;Arg183Val; Arg183Trp; Arg183Tyr; Asn184Ala; Asn184Cys; Asn184Asp;Asn184Glu; Asn184Phe; Asn184Gly; Asn184His; Asn184Ile; Asn184Lys;Asn184Leu; Asn184Met; Asn184Pro; Asn184Gln; Asn184Arg; Asn184Ser;Asn184Thr; Asn184Val; Asn184Trp; Asn184Tyr; Thr185Ala; Thr185Cys;Thr185Asp; Thr185Glu; Thr185Phe; Thr185Gly; Thr185His; Thr185Ile;Thr185Lys; Thr185Leu; Thr185Met; Thr185Asn; Thr185Pro; Thr185Gln;Thr185Arg; Thr185Ser; Thr185Val; Thr185Trp; Thr185Tyr; Thr186Ala;Thr186Cys; Thr186Asp; Thr186Glu; Thr186Phe; Thr186Gly; Thr186His;Thr186Ile; Thr186Lys; Thr186Leu; Thr186Met; Thr186Asn; Thr186Pro;Thr186Gln; Thr186Arg; Thr186Ser; Thr186Val; Thr186Trp; Thr186Tyr;Gly187Ala; Gly187Cys; Gly187Asp; Gly187Glu; Gly187Phe; Gly187His;Gly187Ile; Gly187Lys; Gly187Leu; Gly187Met; Gly187Asn; Gly187Pro;Gly187Gln; Gly187Arg; Gly187Ser; Gly187Thr; Gly187Val; Gly187Trp;Gly187Tyr; Asp188Ala; Asp188Cys; Asp188Glu; Asp188Phe; Asp188Gly;Asp188His; Asp188Ile; Asp188Lys; Asp188Leu; Asp188Met; Asp188Asn;Asp188Pro; Asp188Gln; Asp188Arg; Asp188Ser; Asp188Thr; Asp188Val;Asp188Trp; Asp188Tyr; Ala189Cys; Ala189Asp; Ala189Glu; Ala189Phe;Ala189Gly; Ala189His; Ala189Ile; Ala189Lys; Ala189Leu; Ala189Met;Ala189Asn; Ala189Pro; Ala189Gln; Ala189Arg; Ala189Ser; Ala189Thr;Ala189Val; Ala189Trp; Ala189Tyr; Leu190Ala; Leu190Cys; Leu190Asp;Leu190Glu; Leu190Phe; Leu190Gly; Leu190His; Leu190Ile; Leu190Lys;Leu190Met; Leu190Asn; Leu190Pro; Leu190Gln; Leu190Arg; Leu190Ser;Leu190Thr; Leu190Val; Leu190Trp; Leu190Tyr; Ile191Ala; Ile191Cys;Ile191Asp; Ile191Glu; Ile191Phe; Ile191Gly; Ile191His; Ile191Lys;Ile191Leu; Ile191Met; Ile191Asn; Ile191Pro; Ile191Gln; Ile191Arg;Ile191Ser; Ile191Thr; Ile191Val; Ile191Trp; Ile191Tyr; Arg192Ala;Arg192Cys; Arg192Asp; Arg192Glu; Arg192Phe; Arg192Gly; Arg192His;Arg192Ile; Arg192Lys; Arg192Leu; Arg192Met; Arg192Asn; Arg192Pro;Arg192Gln; Arg192Ser; Arg192Thr; Arg192Val; Arg192Trp; Arg192Tyr;Ala193Cys; Ala193Asp; Ala193Glu; Ala193Phe; Ala193Gly; Ala193His;Ala193Ile; Ala193Lys; Ala193Leu; Ala193Met; Ala193Asn; Ala193Pro;Ala193Gln; Ala193Arg; Ala193Ser; Ala193Thr; Ala193Val; Ala193Trp;Ala193Tyr; Gly194Ala; Gly194Cys; Gly194Asp; Gly194Glu; Gly194Phe;Gly194His; Gly194Ile; Gly194Lys; Gly194Leu; Gly194Met; Gly194Asn;Gly194Pro; Gly194Gln; Gly194Arg; Gly194Ser; Gly194Thr; Gly194Val;Gly194Trp; Gly194Tyr; Val195Ala; Val195Cys; Val195Asp; Val195Glu;Val195Phe; Val195Gly; Val195His; Val195Ile; Val195Lys; Val195Leu;Val195Met; Val195Asn; Val195Pro; Val195Gln; Val195Arg; Val195Ser;Val195Thr; Val195Trp; Val195Tyr; Pro196Ala; Pro196Cys; Pro196Asp;Pro196Glu; Pro196Phe; Pro196Gly; Pro196His; Pro196Ile; Pro196Lys;Pro196Leu; Pro196Met; Pro196Asn; Pro196Gln; Pro196Arg; Pro196Ser;Pro196Thr; Pro196Val; Pro196Trp; Pro196Tyr; Asp197Ala; Asp197Cys;Asp197Glu; Asp197Phe; Asp197Gly; Asp197His; Asp197Ile; Asp197Lys;Asp197Leu; Asp197Met; Asp197Asn; Asp197Pro; Asp197Gln; Asp197Arg;Asp197Ser; Asp197Thr; Asp197Val; Asp197Trp; Asp197Tyr; Gly198Ala;Gly198Cys; Gly198Asp; Gly198Glu; Gly198Phe; Gly198His; Gly198Ile;Gly198Lys; Gly198Leu; Gly198Met; Gly198Asn; Gly198Pro; Gly198Gln;Gly198Arg; Gly198Ser; Gly198Thr; Gly198Val; Gly198Trp; Gly198Tyr;Trp199Ala; Trp199Cys; Trp199Asp; Trp199Glu; Trp199Phe; Trp199Gly;Trp199His; Trp199Ile; Trp199Lys; Trp199Leu; Trp199Met; Trp199Asn;Trp199Pro; Trp199Gln; Trp199Arg; Trp199Ser; Trp199Thr; Trp199Val;Trp199Tyr; Glu200Ala; Glu200Cys; Glu200Asp; Glu200Phe; Glu200Gly;Glu200His; Glu200Ile; Glu200Lys; Glu200Leu; Glu200Met; Glu200Asn;Glu200Pro; Glu200Gln; Glu200Arg; Glu200Ser; Glu200Thr; Glu200Val;Glu200Trp; Glu200Tyr; Val201Ala; Val201Cys; Val201Asp; Val201Glu;Val201Phe; Val201Gly; Val201His; Val201Ile; Val201Lys; Val201Leu;Val201Met; Val201Asn; Val201Pro; Val201Gln; Val201Arg; Val201Ser;Val201Thr; Val201Trp; Val201Tyr; Ala202Cys; Ala202Asp; Ala202Glu;Ala202Phe; Ala202Gly; Ala202His; Ala202Ile; Ala202Lys; Ala202Leu;Ala202Met; Ala202Asn; Ala202Pro; Ala202Gln; Ala202Arg; Ala202Ser;Ala202Thr; Ala202Val; Ala202Trp; Ala202Tyr; Asp203Ala; Asp203Cys;Asp203Glu; Asp203Phe; Asp203Gly; Asp203His; Asp203Ile; Asp203Lys;Asp203Leu; Asp203Met; Asp203Asn; Asp203Pro; Asp203Gln; Asp203Arg;Asp203Ser; Asp203Thr; Asp203Val; Asp203Trp; Asp203Tyr; Lys204Ala;Lys204Cys; Lys204Asp; Lys204Glu; Lys204Phe; Lys204Gly; Lys204His;Lys204Ile; Lys204Leu; Lys204Met; Lys204Asn; Lys204Pro; Lys204Gln;Lys204Arg; Lys204Ser; Lys204Thr; Lys204Val; Lys204Trp; Lys204Tyr;Thr205Ala; Thr205Cys; Thr205Asp; Thr205Glu; Thr205Phe; Thr205Gly;Thr205His; Thr205Ile; Thr205Lys; Thr205Leu; Thr205Met; Thr205Asn;Thr205Pro; Thr205Gln; Thr205Arg; Thr205Ser; Thr205Val; Thr205Trp;Thr205Tyr; Gly206Ala; Gly206Cys; Gly206Asp; Gly206Glu; Gly206Phe;Gly206His; Gly206Ile; Gly206Lys; Gly206Leu; Gly206Met; Gly206Asn;Gly206Pro; Gly206Gln; Gly206Arg; Gly206Ser; Gly206Thr; Gly206Val;Gly206Trp; Gly206Tyr; Ala207Cys; Ala207Asp; Ala207Glu; Ala207Phe;Ala207Gly; Ala207His; Ala207Ile; Ala207Lys; Ala207Leu; Ala207Met;Ala207Asn; Ala207Pro; Ala207Gln; Ala207Arg; Ala207Ser; Ala207Thr;Ala207Val; Ala207Trp; Ala207Tyr; Ala208Cys; Ala208Asp; Ala208Glu;Ala208Phe; Ala208Gly; Ala208His; Ala208Ile; Ala208Lys; Ala208Leu;Ala208Met; Ala208Asn; Ala208Pro; Ala208Gln; Ala208Arg; Ala208Ser;Ala208Thr; Ala208Val; Ala208Trp; Ala208Tyr; Ser209Ala; Ser209Cys;Ser209Asp; Ser209Glu; Ser209Phe; Ser209Gly; Ser209His; Ser209Ile;Ser209Lys; Ser209Leu; Ser209Met; Ser209Asn; Ser209Pro; Ser209Gln;Ser209Arg; Ser209Thr; Ser209Val; Ser209Trp; Ser209Tyr; Tyr210Ala;Tyr210Cys; Tyr210Asp; Tyr210Glu; Tyr210Phe; Tyr210Gly; Tyr210His;Tyr210Ile; Tyr210Lys; Tyr210Leu; Tyr210Met; Tyr210Asn; Tyr210Pro;Tyr210Gln; Tyr210Arg; Tyr210Ser; Tyr210Thr; Tyr210Val; Tyr210Trp;Gly211Ala; Gly211Cys; Gly211Asp; Gly211Glu; Gly211Phe; Gly211His;Gly211Ile; Gly211Lys; Gly211Leu; Gly211Met; Gly211Asn; Gly211Pro;Gly211Gln; Gly211Arg; Gly211Ser; Gly211Thr; Gly211Val; Gly211Trp;Gly211Tyr; Thr212Ala; Thr212Cys; Thr212Asp; Thr212Glu; Thr212Phe;Thr212Gly; Thr212His; Thr212Ile; Thr212Lys; Thr212Leu; Thr212Met;Thr212Asn; Thr212Pro; Thr212Gln; Thr212Arg; Thr212Ser; Thr212Val;Thr212Trp; Thr212Tyr; Arg213Ala; Arg213Cys; Arg213Asp; Arg213Glu;Arg213Phe; Arg213Gly; Arg213His; Arg213Ile; Arg213Lys; Arg213Leu;Arg213Met; Arg213Asn; Arg213Pro; Arg213Gln; Arg213Ser; Arg213Thr;Arg213Val; Arg213Trp; Arg213Tyr; Asn214Ala; Asn214Cys; Asn214Asp;Asn214Glu; Asn214Phe; Asn214Gly; Asn214His; Asn214Ile; Asn214Lys;Asn214Leu; Asn214Met; Asn214Pro; Asn214Gln; Asn214Arg; Asn214Ser;Asn214Thr; Asn214Val; Asn214Trp; Asn214Tyr; Asp215Ala; Asp215Cys;Asp215Glu; Asp215Phe; Asp215Gly; Asp215His; Asp215Ile; Asp215Lys;Asp215Leu; Asp215Met; Asp215Asn; Asp215Pro; Asp215Gln; Asp215Arg;Asp215Ser; Asp215Thr; Asp215Val; Asp215Trp; Asp215Tyr; Ile216Ala;Ile216Cys; Ile216Asp; Ile216Glu; Ile216Phe; Ile216Gly; Ile216His;Ile216Lys; Ile216Leu; Ile216Met; Ile216Asn; Ile216Pro; Ile216Gln;Ile216Arg; Ile216Ser; Ile216Thr; Ile216Val; Ile216Trp; Ile216Tyr;Ala217Cys; Ala217Asp; Ala217Glu; Ala217Phe; Ala217Gly; Ala217His;Ala217Ile; Ala217Lys; Ala217Leu; Ala217Met; Ala217Asn; Ala217Pro;Ala217Gln; Ala217Arg; Ala217Ser; Ala217Thr; Ala217Val; Ala217Trp;Ala217Tyr; Ile218Ala; Ile218Cys; Ile218Asp; Ile218Glu; Ile218Phe;Ile218Gly; Ile218His; Ile218Lys; Ile218Leu; Ile218Met; Ile218Asn;Ile218Pro; Ile218Gln; Ile218Arg; Ile218Ser; Ile218Thr; Ile218Val;Ile218Trp; Ile218Tyr; Ile219Ala; Ile219Cys; Ile219Asp; Ile219Glu;Ile219Phe; Ile219Gly; Ile219His; Ile219Lys; Ile219Leu; Ile219Met;Ile219Asn; Ile219Pro; Ile219Gln; Ile219Arg; Ile219Ser; Ile219Thr;Ile219Val; Ile219Trp; Ile219Tyr; Trp220Ala; Trp220Cys; Trp220Asp;Trp220Glu; Trp220Phe; Trp220Gly; Trp220His; Trp220Ile; Trp220Lys;Trp220Leu; Trp220Met; Trp220Asn; Trp220Pro; Trp220Gln; Trp220Arg;Trp220Ser; Trp220Thr; Trp220Val; Trp220Tyr; Pro221Ala; Pro221Cys;Pro221Asp; Pro221Glu; Pro221Phe; Pro221Gly; Pro221H is; Pro221Ile;Pro221Lys; Pro221Leu; Pro221Met; Pro221Asn; Pro221Gln; Pro221Arg;Pro221Ser; Pro221Thr; Pro221Val; Pro221Trp; Pro221Tyr; Pro222Ala;Pro222Cys; Pro222Asp; Pro222Glu; Pro222Phe; Pro222Gly; Pro222His;Pro222Ile; Pro222Lys; Pro222Leu; Pro222Met; Pro222Asn; Pro222Gln;Pro222Arg; Pro222Ser; Pro222Thr; Pro222Val; Pro222Trp; Pro222Tyr;Lys223Ala; Lys223Cys; Lys223Asp; Lys223Glu; Lys223Phe; Lys223Gly;Lys223His; Lys223Ile; Lys223Leu; Lys223Met; Lys223Asn; Lys223Pro;Lys223Gln; Lys223Arg; Lys223Ser; Lys223Thr; Lys223Val; Lys223Trp;Lys223Tyr; Gly224Ala; Gly224Cys; Gly224Asp; Gly224Glu; Gly224Phe;Gly224His; Gly224Ile; Gly224Lys; Gly224Leu; Gly224Met; Gly224Asn;Gly224Pro; Gly224Gln; Gly224Arg; Gly224Ser; Gly224Thr; Gly224Val;Gly224Trp; Gly224Tyr; Asp225Ala; Asp225Cys; Asp225Glu; Asp225Phe;Asp225Gly; Asp225His; Asp225Ile; Asp225Lys; Asp225Leu; Asp225Met;Asp225Asn; Asp225Pro; Asp225Gln; Asp225Arg; Asp225Ser; Asp225Thr;Asp225Val; Asp225Trp; Asp225Tyr; Pro226Ala; Pro226Cys; Pro226Asp;Pro226Glu; Pro226Phe; Pro226Gly; Pro226His; Pro226Ile; Pro226Lys;Pro226Leu; Pro226Met; Pro226Asn; Pro226Gln; Pro226Arg; Pro226Ser;Pro226Thr; Pro226Val; Pro226Trp; Pro226Tyr; Val227Ala; Val227Cys;Val227Asp; Val227Glu; Val227Phe; Val227Gly; Val227His; Val227Ile;Val227Lys; Val227Leu; Val227Met; Val227Asn; Val227Pro; Val227Gln;Val227Arg; Val227Ser; Val227Thr; Val227Trp; Val227Tyr; Val228Ala;Val228Cys; Val228Asp; Val228Glu; Val228Phe; Val228Gly; Val228His;Val228Ile; Val228Lys; Val228Leu; Val228Met; Val228Asn; Val228Pro;Val228Gln; Val228Arg; Val228Ser; Val228Thr; Val228Trp; Val228Tyr;Leu229Ala; Leu229Cys; Leu229Asp; Leu229Glu; Leu229Phe; Leu229Gly;Leu229His; Leu229Ile; Leu229Lys; Leu229Met; Leu229Asn; Leu229Pro;Leu229Gln; Leu229Arg; Leu229Ser; Leu229Thr; Leu229Val; Leu229Trp;Leu229Tyr; Ala230Cys; Ala230Asp; Ala230Glu; Ala230Phe; Ala230Gly;Ala230His; Ala230Ile; Ala230Lys; Ala230Leu; Ala230Met; Ala230Asn;Ala230Pro; Ala230Gln; Ala230Arg; Ala230Ser; Ala230Thr; Ala230Val;Ala230Trp; Ala230Tyr; Val231Ala; Val231Cys; Val231Asp; Val231Glu;Val231Phe; Val231Gly; Val231His; Val231Ile; Val231Lys; Val231Leu;Val231Met; Val231Asn; Val231Pro; Val231Gln; Val231Arg; Val231Ser;Val231Thr; Val231Trp; Val231Tyr; Leu232Ala; Leu232Cys; Leu232Asp;Leu232Glu; Leu232Phe; Leu232Gly; Leu232His; Leu232Ile; Leu232Lys;Leu232Met; Leu232Asn; Leu232Pro; Leu232Gln; Leu232Arg; Leu232Ser;Leu232Thr; Leu232Val; Leu232Trp; Leu232Tyr; Ser233Ala; Ser233Cys;Ser233Asp; Ser233Glu; Ser233Phe; Ser233Gly; Ser233His; Ser233Ile;Ser233Lys; Ser233Leu; Ser233Met; Ser233Asn; Ser233Pro; Ser233Gln;Ser233Arg; Ser233Thr; Ser233Val; Ser233Trp; Ser233Tyr; Ser234Ala;Ser234Cys; Ser234Asp; Ser234Glu; Ser234Phe; Ser234Gly; Ser234His;Ser234Ile; Ser234Lys; Ser234Leu; Ser234Met; Ser234Asn; Ser234Pro;Ser234Gln; Ser234Arg; Ser234Thr; Ser234Val; Ser234Trp; Ser234Tyr;Arg235Ala; Arg235Cys; Arg235Asp; Arg235Glu; Arg235Phe; Arg235Gly;Arg235His; Arg235Ile; Arg235Lys; Arg235Leu; Arg235Met; Arg235Asn;Arg235Pro; Arg235Gln; Arg235Ser; Arg235Thr; Arg235Val; Arg235Trp;Arg235Tyr; Asp236Ala; Asp236Cys; Asp236Glu; Asp236Phe; Asp236Gly;Asp236His; Asp236Ile; Asp236Lys; Asp236Leu; Asp236Met; Asp236Asn;Asp236Pro; Asp236Gln; Asp236Arg; Asp236Ser; Asp236Thr; Asp236Val;Asp236Trp; Asp236Tyr; Lys237Ala; Lys237Cys; Lys237Asp; Lys237Glu;Lys237Phe; Lys237Gly; Lys237His; Lys237Ile; Lys237Leu; Lys237Met;Lys237Asn; Lys237Pro; Lys237Gln; Lys237Arg; Lys237Ser; Lys237Thr;Lys237Val; Lys237Trp; Lys237Tyr; Lys238Ala; Lys238Cys; Lys238Asp;Lys238Glu; Lys238Phe; Lys238Gly; Lys238His; Lys238Ile; Lys238Leu;Lys238Met; Lys238Asn; Lys238Pro; Lys238Gln; Lys238Arg; Lys238Ser;Lys238Thr; Lys238Val; Lys238Trp; Lys238Tyr; Asp239Ala; Asp239Cys;Asp239Glu; Asp239Phe; Asp239Gly; Asp239His; Asp239Ile; Asp239Lys;Asp239Leu; Asp239Met; Asp239Asn; Asp239Pro; Asp239Gln; Asp239Arg;Asp239Ser; Asp239Thr; Asp239Val; Asp239Trp; Asp239Tyr; Ala240Cys;Ala240Asp; Ala240Glu; Ala240Phe; Ala240Gly; Ala240His; Ala240Ile;Ala240Lys; Ala240Leu; Ala240Met; Ala240Asn; Ala240Pro; Ala240Gln;Ala240Arg; Ala240Ser; Ala240Thr; Ala240Val; Ala240Trp; Ala240Tyr;Lys241Ala; Lys241Cys; Lys241Asp; Lys241Glu; Lys241Phe; Lys241Gly;Lys241His; Lys241Ile; Lys241Leu; Lys241Met; Lys241Asn; Lys241Pro;Lys241Gln; Lys241Arg; Lys241Ser; Lys241Thr; Lys241Val; Lys241Trp;Lys241Tyr; Tyr242Ala; Tyr242Cys; Tyr242Asp; Tyr242Glu; Tyr242Phe;Tyr242Gly; Tyr242His; Tyr242Ile; Tyr242Lys; Tyr242Leu; Tyr242Met;Tyr242Asn; Tyr242Pro; Tyr242Gln; Tyr242Arg; Tyr242Ser; Tyr242Thr;Tyr242Val; Tyr242Trp; Asp243Ala; Asp243Cys; Asp243Glu; Asp243Phe;Asp243Gly; Asp243His; Asp243Ile; Asp243Lys; Asp243Leu; Asp243Met;Asp243Asn; Asp243Pro; Asp243Gln; Asp243Arg; Asp243Ser; Asp243Thr;Asp243Val; Asp243Trp; Asp243Tyr; Asp244Ala; Asp244Cys; Asp244Glu;Asp244Phe; Asp244Gly; Asp244His; Asp244Ile; Asp244Lys; Asp244Leu;Asp244Met; Asp244Asn; Asp244Pro; Asp244Gln; Asp244Arg; Asp244Ser;Asp244Thr; Asp244Val; Asp244Trp; Asp244Tyr; Lys245Ala; Lys245Cys;Lys245Asp; Lys245Glu; Lys245Phe; Lys245Gly; Lys245His; Lys245Ile;Lys245Leu; Lys245Met; Lys245Asn; Lys245Pro; Lys245Gln; Lys245Arg;Lys245Ser; Lys245Thr; Lys245Val; Lys245Trp; Lys245Tyr; Leu246Ala;Leu246Cys; Leu246Asp; Leu246Glu; Leu246Phe; Leu246Gly; Leu246His;Leu246Ile; Leu246Lys; Leu246Met; Leu246Asn; Leu246Pro; Leu246Gln;Leu246Arg; Leu246Ser; Leu246Thr; Leu246Val; Leu246Trp; Leu246Tyr;Ile247Ala; Ile247Cys; Ile247Asp; Ile247Glu; Ile247Phe; Ile247Gly;Ile247His; Ile247Lys; Ile247Leu; Ile247Met; Ile247Asn; Ile247Pro;Ile247Gln; Ile247Arg; Ile247Ser; Ile247Thr; Ile247Val; Ile247Trp;Ile247Tyr; Ala248Cys; Ala248Asp; Ala248Glu; Ala248Phe; Ala248Gly;Ala248His; Ala248Ile; Ala248Lys; Ala248Leu; Ala248Met; Ala248Asn;Ala248Pro; Ala248Gln; Ala248Arg; Ala248Ser; Ala248Thr; Ala248Val;Ala248Trp; Ala248Tyr; Glu249Ala; Glu249Cys; Glu249Asp; Glu249Phe;Glu249Gly; Glu249His; Glu249Ile; Glu249Lys; Glu249Leu; Glu249Met;Glu249Asn; Glu249Pro; Glu249Gln; Glu249Arg; Glu249Ser; Glu249Thr;Glu249Val; Glu249Trp; Glu249Tyr; Ala250Cys; Ala250Asp; Ala250Glu;Ala250Phe; Ala250Gly; Ala250His; Ala250Ile; Ala250Lys; Ala250Leu;Ala250Met; Ala250Asn; Ala250Pro; Ala250Gln; Ala250Arg; Ala250Ser;Ala250Thr; Ala250Val; Ala250Trp; Ala250Tyr; Thr251Ala; Thr251Cys;Thr251Asp; Thr251Glu; Thr251Phe; Thr251Gly; Thr251His; Thr251Ile;Thr251Lys; Thr251Leu; Thr251Met; Thr251Asn; Thr251Pro; Thr251Gln;Thr251Arg; Thr251Ser; Thr251Val; Thr251Trp; Thr251Tyr; Lys252Ala;Lys252Cys; Lys252Asp; Lys252Glu; Lys252Phe; Lys252Gly; Lys252His;Lys252Ile; Lys252Leu; Lys252Met; Lys252Asn; Lys252Pro; Lys252Gln;Lys252Arg; Lys252Ser; Lys252Thr; Lys252Val; Lys252Trp; Lys252Tyr;Val253Ala; Val253Cys; Val253Asp; Val253Glu; Val253Phe; Val253Gly;Val253His; Val253Ile; Val253Lys; Val253Leu; Val253Met; Val253Asn;Val253Pro; Val253Gln; Val253Arg; Val253Ser; Val253Thr; Val253Trp;Val253Tyr; Val254Ala; Val254Cys; Val254Asp; Val254Glu; Val254Phe;Val254Gly; Val254His; Val254Ile; Val254Lys; Val254Leu; Val254Met;Val254Asn; Val254Pro; Val254Gln; Val254Arg; Val254Ser; Val254Thr;Val254Trp; Val254Tyr; Met255Ala; Met255Cys; Met255Asp; Met255Glu;Met255Phe; Met255Gly; Met255His; Met255Ile; Met255Lys; Met255Leu;Met255Asn; Met255Pro; Met255Gln; Met255Arg; Met255Ser; Met255Thr;Met255Val; Met255Trp; Met255Tyr; Lys256Ala; Lys256Cys; Lys256Asp;Lys256Glu; Lys256Phe; Lys256Gly; Lys256His; Lys256Ile; Lys256Leu;Lys256Met; Lys256Asn; Lys256Pro; Lys256Gln; Lys256Arg; Lys256Ser;Lys256Thr; Lys256Val; Lys256Trp; Lys256Tyr; Ala257Cys; Ala257Asp;Ala257Glu; Ala257Phe; Ala257Gly; Ala257His; Ala257Ile; Ala257Lys;Ala257Leu; Ala257Met; Ala257Asn; Ala257Pro; Ala257Gln; Ala257Arg;Ala257Ser; Ala257Thr; Ala257Val; Ala257Trp; Ala257Tyr; Leu258Ala;Leu258Cys; Leu258Asp; Leu258Glu; Leu258Phe; Leu258Gly; Leu258His;Leu258Ile; Leu258Lys; Leu258Met; Leu258Asn; Leu258Pro; Leu258Gln;Leu258Arg; Leu258Ser; Leu258Thr; Leu258Val; Leu258Trp; Leu258Tyr;Asn259Ala; Asn259Cys; Asn259Asp; Asn259Glu; Asn259Phe; Asn259Gly;Asn259His; Asn259Ile; Asn259Lys; Asn259Leu; Asn259Met; Asn259Pro;Asn259Gln; Asn259Arg; Asn259Ser; Asn259Thr; Asn259Val; Asn259Trp;Asn259Tyr; Met260Ala; Met260Cys; Met260Asp; Met260Glu; Met260Phe;Met260Gly; Met260His; Met260Ile; Met260Lys; Met260Leu; Met260Asn;Met260Pro; Met260Gln; Met260Arg; Met260Ser; Met260Thr; Met260Val;Met260Trp; Met260Tyr; Asn261Ala; Asn261Cys; Asn261Asp; Asn261Glu;Asn261Phe; Asn261Gly; Asn261His; Asn261Ile; Asn261Lys; Asn261Leu;Asn261Met; Asn261Pro; Asn261Gln; Asn261Arg; Asn261Ser; Asn261Thr;Asn261Val; Asn261Trp; Asn261Tyr; Gly262Ala; Gly262Cys; Gly262Asp;Gly262Glu; Gly262Phe; Gly262His; Gly262Ile; Gly262Lys; Gly262Leu;Gly262Met; Gly262Asn; Gly262Pro; Gly262Gln; Gly262Arg; Gly262Ser;Gly262Thr; Gly262Val; Gly262Trp; Gly262Tyr; Lys263Ala; Lys263Cys;Lys263Asp; Lys263Glu; Lys263Phe; Lys263Gly; Lys263His; Lys263Ile;Lys263Leu; Lys263Met; Lys263Asn; Lys263Pro; Lys263Gln; Lys263Arg;Lys263Ser; Lys263Thr; Lys263Val; Lys263Trp; Lys263Tyr; Met 264Ala; Met264Cys; Met 264Asp; Met 264Glu; Met 264Phe; Met 264Gly; Met 264His; Met264Ile; Met 264Lys; Met 264Leu; Met 264Asn; Met 264Pro; Met 264Gln; Met264Arg; Met 264Ser; Met 264Thr; Met 264Val; Met 264Trp; Met 264Tyr; Asn265Ala; Asn 265Cys; Asn 265Asp; Asn 265Glu; Asn 265Phe; Asn 265Gly; Asn265His; Asn 265Ile; Asn 265Lys; Asn 265Leu; Asn 265Met; Asn 265Pro; Asn265Gln; Asn 265Arg; Asn 265Ser; Asn 265Thr; Asn 265Val; Asn 265Trp; Asn265Tyr; Gly 266Ala; Gly 266Cys; Gly 266Asp; Gly 266Glu; Gly 266Phe; Gly266His; Gly 266Ile; Gly 266Lys; Gly 266Leu; Gly 266Met; Gly 266Asn; Gly266Pro; Gly 266Gln; Gly 266Arg; Gly 266Ser; Gly 266Thr; Gly 266Val; Gly266Trp; Gly 266Tyr; Lys267Ala; Lys267Cys; Lys267Asp; Lys267Glu;Lys267Phe; Lys267Gly; Lys267His; Lys267Ile; Lys267Leu; Lys267Met;Lys267Asn; Lys267Pro; Lys267Gln; Lys267Arg; Lys267Ser; Lys267Thr;Lys267Val; Lys267Trp; and Lys267Tyr. In some embodiments, SEQ ID NO: 1may have a Met and/or Thr preceeding the first residue of the sequence.These residues may be similarly mutated as above.

In all of these mutants, the numbering of residues corresponds to SEQ IDNO: 1. These residue numbers may be converted to Ambler numbers (Ambleret al., 1991, A standard numbering scheme for the Class A β-lactamases,Biochem. J. 276:269-272, the contents of which are hereby incorporatedby reference) through use of any conventional bioinformatic method, forexample by using BLAST (Basic Local Alignment Search Tools) or FASTA(FAST-All). For example, residue 244 corresponds to Ambler 276. Forexample, the following conversions may be used:

Ambler Classification No. SEQ ID NO: 1 Residue F33 F6 I72 I44 Q135 Q105G156 G126 T160 T130 A232 A202 A237 A207 A238 A208 S240 S209 T243 T212R244 R213 S266 S234 D276 D244

Furthermore, percent identity may also be assessed with theseconventional bioinformatic methods.

In one aspect, the beta-lactamase polypeptide produced by methods of theinvention comprises an amino acid sequence having at least about 60%(e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%,or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%)sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and one or more ofthe following mutations of Ambler classification: F33X, Q135X, G156X,A232X, A237X, A238X, S240X, T243X, R244X, S266X, and D276X, wherein X isany naturally-occurring amino acid. In some embodiments, X is anaturally occurring hydrophilic or hydrophobic amino acid residue or anon-classical amino acid.

In another aspect, the beta-lactamase polypeptide produced by methods ofthe invention comprises an amino acid sequence having at least 60%sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and one or more ofthe following mutations of Ambler classification: a hydrophobic residueother than phenylalanine (F) at position 33; a hydrophobic residue otherthan glutamine (Q) at position 135; a hydrophilic residue other thanglycine (G) at position 156; a hydrophobic residue other than alanine(A) at position 232; a hydrophilic residue other than alanine (A) atposition 237; a hydrophobic or hydrophilic residue other than alanine(A) at position 238; a hydrophilic residue other than serine (S) atposition 240; a hydrophobic residue other than threonine (T) at position243; a hydrophobic residue other than arginine (R) at position 244; ahydrophilic residue other than serine (S) at position 266; and ahydrophilic residue other than aspartate (D) at position 276.

As used throughout, a hydrophilic amino acid residue may include a polarand positively charged hydrophilic residue selected from arginine (R)and lysine (K), a polar and neutral of charge hydrophilic residueselected from asparagine (N), glutamine (Q), serine (S), threonine (T),proline (P), and cysteine (C), a polar and negatively chargedhydrophilic residue selected from aspartate (D) and glutamate (E), or anaromatic, polar and positively charged hydrophilic including histidine(H). As used throughout, a hydrophobic amino acid residue may include ahydrophobic, aliphatic amino acid selected from glycine (G), alanine(A), leucine (L), isoleucine (I), methionine (M), or valine (V) or ahydrophobic, aromatic amino acid selected from phenylalanine (F),tryptophan (W), or tyrosine (Y).

Mutations may be made to the gene sequence of a beta-lactamase (e.g. SEQID NOs: 2 and 4) by reference to the genetic code, including taking intoaccount codon degeneracy.

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises one or more of the following mutations atpositions of Ambler classification: F33Y, Q135M, G156R, A232G, A237S,A238G or T, S240P or D, T243I, R244T, S266N, D276N or R or K. In oneembodiment, the beta-lactamases and/or pharmaceutical compositionscomprise Q135M. In another embodiment, the beta-lactamases and/orpharmaceutical compositions comprise G156R and A238T. In anotherembodiment, the beta-lactamases and/or pharmaceutical compositionscomprise F33Y and D276N. In still another embodiment, thebeta-lactamases and/or pharmaceutical compositions comprise F33Y, S240P,and D276N. In one embodiment, the beta-lactamases and/or pharmaceuticalcompositions comprise F33Y, A238T, and D276N. In another embodiment, thebeta-lactamases and/or pharmaceutical compositions comprise A232G,A237S, A238G, and S240D. In a further embodiment, the beta-lactamasesand/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D,and R244T. In another embodiment, the beta-lactamases and/orpharmaceutical compositions comprise A232G, A237S, A238G, S240D, andD276R. In one embodiment, the beta-lactamases and/or pharmaceuticalcompositions comprise A232G, A237S, A238G, S240D, and D276K. In oneembodiment, the beta-lactamases and/or pharmaceutical compositionscomprise A232G, A237S, A238G, S240D, and Q135M. In one embodiment, thebeta-lactamases and/or pharmaceutical compositions comprise A238T. Inone embodiment, the beta-lactamases and/or pharmaceutical compositionscomprise T243I, S266N, and D276N. In one embodiment, the beta-lactamasesand/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D,and D276N.

In various embodiments, the beta-lactamase polypeptide produced bymethods of the invention comprises one or more of the followingmutations:

Mutations relative to P1A (based on the Ambler classification) Name Wildtype RS310 (or P1A) D276N IS118 (or P3A) I72S IS222 T160F IS203 R244TIS217 R244T D276K IS215 Q135M IS197 G156R A238T IS235 F33Y D276N IS158F33Y S240P D276N IS230 (or IS181) F33Y A238T D276N IS232 (or IS180) I72SQ135M T160F (Block 1 mutants) IS227 A232G A237S A238G S240D (Block 2mutants) IS191 A232G A237S A238G S240D R244T IS229 A232G A237S A238GS240D D276R IS219 A232G A237S A238G S240D D276K IS221 A232G A237S A238GS240D Q135M IS224 A238T IS233 T243I S266N D276N IS234 (or IS176) A232GA237S A238G S240D D276N IS288 (or P4A)

In various embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 60%sequence identity with one or more of the mutants provided in the tabledirectly above.

In illustrative embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 60%sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and the following ofAmbler classification: a residue other than aspartate (D) at position276.

In illustrative embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 90%, or95%, or 97%, or 99% sequence identity with SEQ ID NO: 1 and ahydrophilic amino acid residue other than aspartic acid (D) at aposition corresponding to position 276 according to Amblerclassification, wherein: the hydrophilic amino acid residue isasparagine (N) and the beta-lactamase hydrolyzes ceftriaxonesubstantially more efficiently than a beta-lactamase of SEQ ID NO: 1that has an aspartic acid (D) at a position corresponding to position276 according to Ambler classification.

In illustrative embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 90%, or95%, or 97%, or 99% sequence identity with SEQ ID NO: 1 and ahydrophilic amino acid residue other than aspartic acid (D) at aposition corresponding to position 276 according to Amblerclassification, wherein: the hydrophilic amino acid residue is arginine(R) and the beta-lactamase hydrolyzes ceftriaxone substantially moreefficiently than a beta-lactamase of SEQ ID NO: 1 that has an asparticacid (D) at a position corresponding to position 276 according to Amblerclassification.

In some embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 90%, or95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 5, i.e.P3A:

SEQ ID NO: 5 TEMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVADKTGAASYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDNKLIAEATKVVMKALNMNGK.

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises an amino acid sequence having at least about60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%) sequence identity with SEQ ID NO: 5.

An illustrative polynucleotide of the invention is SEQ ID NO: 6, whichis the full nucleotide sequence of P3A:

SEQ ID NO: 6: atgactgagatgaaagatgattttgcgaagctggaagaacagtttgacgcaaaattgggcattttcgcgttggacacgggtacgaatcgtacggttgcctaccgtccggacgagcgcttcgccttcgcgagcacgatcaaagccctgaccgtcggcgtgctgctccagcaaaagagcatcgaggacctgaaccagcgcattacctacacccgtgatgatctggtgaactataatccgatcaccgagaaacacgttgataccggtatgaccctgaaagaactggcagatgcaagcctgcgctacagcgataacgcggctcagaatctgattctgaagcaaatcggtggtccggagagcttgaagaaagaactgcgtaaaatcggcgatgaagtcactaatccggagcgttttgagccggagctgaacgaagtgaatccgggtgaaacgcaagacacgagcaccgcgcgtgcgcttgtcacctccctgcgcgctttcgcactggaagataagctgccgtcggagaaacgcgagctgctgatcgactggatgaagcgcaatacgaccggcgacgcgctgattcgtgcgggcgttccggacggttgggaagtggctgacaagaccggtgcggcgagctacggcacccgtaacgatatcgcgatcatttggccacctaaaggtgacccggtcgtgctggccgtactgagcagccgtgacaagaaagacgcaaagtatgataacaagctgattgcagaggcgaccaaagttgttatgaaggcactgaacatgaatggtaag

In some embodiments, the polynucleotide of the present invention has atleast about 60% (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99%) sequence identity with SEQ ID NO: 6.

In illustrative embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 60%sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and the following ofAmbler classification: a hydrophobic residue other than alanine (A) atposition 232; a hydrophilic residue other than alanine (A) at position237; a hydrophobic residue other than alanine (A) at position 238; ahydrophilic residue other than serine (S) at position 240; and ahydrophilic residue other than aspartate (D) at position 276. In someembodiments, the hydrophobic residue other than alanine (A) at position232 is glycine (G). In some embodiments, the hydrophilic residue otherthan alanine (A) at position 237 is serine (S). In some embodiments, thehydrophobic residue other than alanine (A) at position 238 is glycine(G). In some embodiments, the hydrophilic residue other than serine (S)at position 240 is aspartate (D). In some embodiments, the other thanaspartate (D) at position 276 is asparagine (N). In some embodiments,the beta-lactamase and/or pharmaceutical composition comprises one ormore of A232G, A237S, A238G, S240D, and D276N. In some embodiments, thebeta-lactamase and/or pharmaceutical composition comprises all of A232G,A237S, A238G, S240D, and D276N, the sequence of which is SEQ ID NO: 7,i.e. P4A. In some embodiments, the beta-lactamase and/or pharmaceuticalcomposition comprises an amino acid sequence having at least 90%, or95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 7.

SEQ ID NO: 7 EMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVGDKTGSGDYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAK YDNKLIAEATKVVMKALNMNGK

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises an amino acid sequence having at least about60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%) sequence identity with SEQ ID NO: 7.

SEQ ID NO: 8, is derived from SEQ ID NO: 7, and further includes thesignal and the addition of the QASKT (SEQ ID NO: 11) amino acids (thecoding region is underlined):

MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAQASKTEMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVGDKTGSGDYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDNKLIAEATK VVMKALNMNGK

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises an amino acid sequence having at least about60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%) sequence identity with SEQ ID NO: 8.

In some embodiments, the beta-lactamase and/or pharmaceuticalcomposition comprises an amino acid sequence having at least 90%, or95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 8.

An illustrative polynucleotide of the invention is SEQ ID NO: 9, whichis the full nucleotide sequence of A232G, A237S, A238G, S240D, and D276Nmutant, Hind III site (AAGCTT-in bold) and additional K and T aminoacids. In some embodiments, the underlined portion of SEQ ID NO: 9, isomitted. The leader and additional nucleotides (Hind III site and K andT amino acids—for the addition of the amino acid sequence QASKT (SEQ IDNO: 11)) are underlined.

atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagcgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtgggtgataaaactggaagcggagattatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttottgcagtattatccagcagggataaaaaggacgccaagtatgataataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, the polynucleotide of the present invention has atleast about 60% (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99%) sequence identity with SEQ ID NO: 9 (with or withoutthe underlined portion).

In various aspects, the beta-lactamases polypeptide has the sequence ofSEQ ID NO: 10 (i.e., P2A) or is derived by one or more mutations of SEQID NO: 10:

ETGTISISQLNKNVWVHTELGYFNGEAVPSNGLVLNTSKGLVLVDSSWDNKLTKELIEMVEKKFQKRVTDVIITHAHADRIGGITALKERGIKAHSTALTAELAKNSGYEEPLGDLQTITSLKFGNTKVETFYPGKGHTEDNIVVWLPQYQILAGGCLVKSAEAKDLGNVADAYVNEWSTSIENVLKRYGNINSVVPGHGEVGDKGLLLHTLDLLK.

In some embodiments, the beta-lactamase polypeptide produced by methodsof the invention comprises an amino acid sequence having at least about60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%) sequence identity with SEQ ID NO: 10.

In some embodiments, the beta-lactamase and/or pharmaceuticalcomposition comprises an amino acid sequence having at least 90%, or95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 10.

Additional sequences of beta-lactamases including P1A, P2A, P3A, and P4Aand derivatives thereof are described for example, in WO 2011/148041 andPCT/US2015/026457, the entire contents of which are hereby incorporatedby reference.

The invention provides for polynucleotides encoding a beta-lactamasepolypeptide, including, for example, vectors, comprising suchpolynucleotides. Such polynucleotides may further comprise, in additionto sequences encoding the beta-lactamases of the invention, one or moreexpression control elements. For example, the polynucleotide, maycomprise one or more promoters or transcriptional enhancers, ribosomalbinding sites, transcription termination signals, and polyadenylationsignals, as expression control elements. In an embodiment, thepolynucleotide includes expression control elements that directexpression of the beta-lactamase in the cytoplasm.

The polynucleotide may be inserted within a suitable vector, which isutilized to transform a suitable host cell such as an E. coli cell forexpression. The vector may be any self-replicating DNA molecule that cantransfer a DNA between host cells, including, for example, a plasmidcloning vector. In some embodiments, the vector can remain episomal orbecome chromosomally integrated, as long as the insert encoding thetherapeutic agent can be transcribed. Vectors can be constructed bystandard recombinant DNA technology. Vectors can be, for example,plasmids, phages, cosmids, phagemids, viruses, or any other types knownin the art, which are used for replication and expression in prokaryoticor eukaryotic cells (e.g. an adenovirus; a retrovirus; a lentivirus; anscAAV; pGEX vector; pET vector; and pHT vector). Exemplary vectors thatmay be used include, for example, the pAVE011 vector. Preparations ofthe pAVE011 vector is described in EP Patent No. 0502637, EP Patent No.2386642, and U.S. Pat. No. 6,537,779, the entire contents of which arehereby incorporated by reference. It will be appreciated by one of skillin the art that a wide variety of components known in the art (such asexpression control elements) may be included in such vectors, includinga wide variety of transcription signals, such as promoters and othersequences that regulate the binding of RNA polymerase onto the promoter.Any promoter known to be effective in E coli cells in which the vectorwill be expressed can be used to initiate expression of the therapeuticagent. In one embodiment, the promoter is effective for directingexpression of the beta-lactamase polypeptide in the cytoplasm. Suitablepromoters may be inducible or constitutive. Examples of suitablepromoters include, for example, the pET system (INVITROGEN), lacpromoter, tac, trc, T7, T7A3 promoter, PhoA, Phage lambda pR, lambda pLpromoter (see, e.g. J Ind Microbiol Biotechnol (2012) 39:383-399; CurrOpin Biotech 2001, 12: 195, the contents of which are herebyincorporated by reference), Pspac, PgroES, Pgsi, Plux and amyQ promoterand/or amyQ signal peptide from Bacillus amyloliquefaciens (by way ofnon-limiting example Gen Bank ID No. J01542.1, the contents of which arehereby incorporated by reference). The promoter may be inducible (e.g.via IPTG, metabolites, temperature). In one embodiment, the cytoplasmicexpression of the beta-lactamase polypeptide is driven by the IPTGinducible Lacl promoter. In one embodiment, cytoplasmic expression ofthe beta-lactamase polypeptide is induced by adding IPTG to thebacterial culture.

In various embodiments, the transformed E. coli cell is grown for a timeunder conditions sufficient to produce cytoplasmic expression of thebeta-lactamase polypeptide. Any type of media that will support growthand reproduction of E. coli cell in cultures is useful for practicingthe method of the invention. After growth of the cultures, the E. colicell is typically lysed using osmotic shock, sonication or otherstandard means, and the expressed beta-lactamase polypeptide is isolatedfrom the soluble fraction. Any protein purification method may beemployed for this purpose, such as dialysis, gel filtration, ionexchange chromatography, affinity chromatography, electrophoresis, or acombination of steps.

In various embodiments, the beta-lactamases produced by methods of theinvention possess functional characteristics that make them desirablefor a variety of uses, including therapeutic uses. Methods ofcharacterizing beta-lactamases are known in the art (e.g. nitrocefinassay as described by O'Callaghan, et al. Antimicrob. Agents Chemother,1:283-288; the various methods of Viswanatha et al. Methods Mol Med.2008; 142:239-60).

In one embodiment, the beta-lactamases produced by methods of theinvention hydrolyze one or more of penicillins and cephalosporins. Asused throughout, penicillins include, for example, Amoxicillin (e.g.NOVAMOX, AMOXIL); Ampicillin (e.g. PRINCIPEN); Azlocillin; Carbenicillin(e.g. GEOCILLIN); Cloxacillin (e.g. TEGOPEN); Dicloxacillin (e.g.DYNAPEN); Flucloxacillin (e.g. FLOXAPEN); Mezlocillin (e.g. MEZLIN);Methicillin (e.g. STAPHCILLIN); Nafcillin (e.g. UNIPEN); Oxacillin (e.g.PROSTAPHLIN); Penicillin G (e.g. PENTIDS or PFIZERPEN); Penicillin V(e.g. VEETIDS (PEN-VEE-K)); Piperacillin (e.g. PIPRACIL); Temocillin(e.g. NEGABAN); and Ticarcillin (e.g. TICAR). As used throughout,cephalosporins include, for example, a first generation cephalosporin(e.g. Cefadroxil (e.g. DURICEF); Cefazolin (e.g. ANCEF); Ceftolozane,Cefalotin/Cefalothin (e.g. KEFLIN); Cefalexin (e.g. KEFLEX); a secondgeneration cephalosporin (e.g. Cefaclor (e.g. DISTACLOR); Cefamandole(e.g. MANDOL); Cefoxitin (e.g. MEFOXIN); Cefprozil (e.g. CEFZIL);Cefuroxime (e.g. CEFTIN, ZINNAT)); a third generation cephalosporin(e.g. Cefixime (e.g. SUPRAX); Cefdinir (e.g. OMNICEF, CEFDIEL);Cefditoren (e.g. SPECTRACEF); Cefoperazone (e.g. CEFOBID); Cefotaxime(e.g. CLAFORAN); Cefpodoxime (e.g. VANTIN); Ceftazidime (e.g. FORTAZ);Ceftibuten (e.g. CEDAX) Ceftizoxime (e.g. CEFIZOX); and Ceftriaxone(e.g. ROCEPHIN)); a fourth generation cephalosporin (e.g. Cefepime (e.g.MAXIPIME)); or a fifth generation cephalosporin (e.g. Ceftarolinefosamil (e.g. TEFLARO); Ceftobiprole (e.g. ZEFTERA)). In a specificembodiment, cephalosporins include, for example, cefoperazone,ceftriaxone or cefazolin. In a specific embodiment, the inventivebeta-lactamases have improved catalytic efficiency againstcephalosporins as compared to SEQ ID NO: 1.

In various embodiments, the beta-lactamases possess desirable enzymekinetic characteristics. For example, in some embodiments, thebeta-lactamases possess a low K_(M) for at least one cephalosporin,including, for example, a K_(M) of less than about 500 μM, or about 100μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100 nM), or about0.01 μM (10 nM), or about 1 nM. For example, in some embodiments, thebeta-lactamases possess a low K_(M) for at least one penicillin,including, for example, a K_(M) of less than about 500 μM, or about 100μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100 nM), or about0.01 μM (10 nM), or about 1 nM. In various embodiments, the inventivebeta-lactamases possess a high V_(max) for at least one cephalosporin,including, for example, V_(max) which is greater than about 100 s-1, orabout 1000 s-1, or about 10000 s-1, or about 100000 s-1, or about1000000 s-1. In various embodiments, the inventive beta-lactamasespossess a high V_(max) for at least one penicillin, including, forexample, V_(max) which is greater than about 100 s-1, or about 1000 s-1,or about 10000 s-1, or about 100000 s-1, or about 1000000 s-1. Invarious embodiments, the inventive beta-lactamases possess catalyticefficiency is greater than about 10⁶ M⁻¹ s⁻¹ for at least onecephalosporin. In various embodiments, the inventive beta-lactamasespossess catalytic efficiency is greater than about 10⁶ M⁻¹ s⁻¹ for atleast one penicillin. In various embodiments, the inventivebeta-lactamases possess the desirable enzyme kinetic characteristics forat least one of either or both of cephalosporins and penicillins.

In various embodiments, the inventive beta-lactamases are stable and/oractive in the GI tract, e.g. in one or more of the mouth, esophagus,stomach, duodenum, small intestine, duodenum, jejunum, ileum, largeintestine, colon transversum, colon descendens, colon ascendens, colonsigmoidenum, cecum, and rectum. In a specific embodiment, thebeta-lactamase is stable in the large intestine, optionally selectedfrom one or more of colon transversum, colon descendens, colonascendens, colon sigmoidenum and cecum. In a specific embodiment, thebeta-lactamase is stable in the small intestine, optionally selectedfrom one or more of duodenum, jejunum, and ileum. In some embodiments,the beta-lactamase is resistant to proteases in the GI tract, includingfor example, the small intestine. In some embodiments, thebeta-lactamase is substantially active at a pH of about 6.0 to about7.5, e.g. about 6.0, or about 6.1, or about 6.2, or about 6.3, or about6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about6.9, or about 7.0, or about 7.1, or about 7.2, or about 7.3, or about7.4, or about 7.5 (including, for example, via formulation, as describedherein). In various embodiments, the beta-lactamases of the presentinvention are resistant to one or more beta-lactamase inhibitors,optionally selected from avibactam, tazobactam, sulbactam, andclavulanic acid. In some embodiments, stable refers to an enzyme thathas a long enough half-life and maintains enough activity fortherapeutic effectiveness.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Production of Beta-Lactamases in Bacillus Strains

P1A-protein was produced by Bacillus subtilis RS310 production strain inapproximately 10,000 liter fed-batch fermentation. The Bacillus subtilisRS310 strain was asporogenic, tryptophan auxotrophic and secretedP1A-protein into the culture broth. Specifically, cell culturing of theP1A-protein comprised two inoculum (1%) expansion stages in shake flasks(WCB vial→100 mL→2×1200 mL) followed by a seed fermentation stage (220L, 2.5%). The main fed-batch fermentation was conducted in approximately10,000 L working volume. The main fermentation was started as batchfermentation with an initial volume of 9,000 L of growth medium. Afterabout 9 hours when most of glucose in the growth medium was consumed,feeding with a feed solution (approx. 1500-2000 L) containing glucoseand phosphate was started. In order to keep glucose at adequate levels(0.5-5 mg/mL) during the feeding phase, predefined feeding profile wasused, which may be adjusted during the process based on glucosemeasurements. The P1A protein was constitutively produced and secretedextracellularly into the culture broth.

During fermentation the critical operational parameters were monitoredand controlled including glucose concentration, pH (7±0.2), dissolvedoxygen level (10-20%), temperature (37±10C) and foam level. Stirringrate was controlled starting with gentle mixing and increasing to amaximum of 138-145 rpm. Air flow into the vessel was adjusted to 0.5-1vvm. Progression of fermentation was monitored by P1A content (enzymeactivity measurement) and cell density measurements (OD 600 nm). Themain fermentation achieved a P1A titer of about 1-1.2 mg/mL (by HPLC)typically after 16-22 hours. The final cell density was typicallyapproximately OD 50 (d.w. 16-17 g/L). After completion of cultivation,the content of fermenter was cooled down to 11±3° C.

After fermentation the cells were removed from P1A-protein containingbroth by continuous centrifugation followed by microfiltration. P1Acontaining filtrate was concentrated by ultrafiltration and P1Aconcentrate was further diafiltered, conditioned and passed through adisposable anion exchange filter cartridge in flow-through mode afterwhich the filtrate was further diafiltered to remove NaCl. This preparedthe solution for the following two stage P1A-protein crystallisationincluding; crystallisation, crystal harvesting, washing and dissolution.Finally, after the second crystallisation step, P1A-protein crystalswere suspended in water and dissolved and final concentration ofP1A-protein solution was adjusted. The protein solution was filtered(0.2 um) to reduce bioburden and finally dispensed into sterile plasticcontainers, frozen and stored at −70° C.

Example 2: Intracellular Gene Design for the Expression of P3Aβ-Lactamase

The purpose of this study was to improve 3-lactamase expression. To doso, the pAVEway™ advance protein expression system was employed in E.coli. P3A was used throughout this study for testing 3-lactamaseexpression. The gene sequence for directing the intracellular expressionof P3A is SEQ ID NO: 6.

The P3A gene was cloned into the pAVEway™ intercellular (cytoplasmic)construct, pAVE011, and the plasmid was verified with PCR and DNAsequencing. The designed P3A expression construct provided a relativelyhomogeneous N-terminus with the N-terminal methionine removed about 95%of the time.

Following construction of the intercellular expression plasmid, theconstruct was transformed in the following E. coli strains: CLD977(W3110 E. coli host) and CLD990 (BL21 E. coli host). After constructionof the 3-lactamase intracellular expression strains, P3A was expressedand characterized as further described in Examples 2 and 3,respectively.

Additionally, the P3A gene was cloned into the pAVEway™ periplasmicconstruct, pAVE029+gene 1 or gene 7 (gene 1 and gene 7 are differentsecretion leaders). Again, the plasmid was verified with PCR and DNAsequencing.

Following construction of the periplasmic expression plasmid, theconstruct was transformed in the following E. coli strains: CLD981 (gene1 leader, W3110 E. coli host) and CLD982 (gene 7 leader, BL21 E. colihost). After construction of the periplasmic 3-lactamase expressionstrains, P3A was expressed and characterized as further described inExamples 2 and 3, respectively.

Example 3: P3A β-Lactamase Fermentation

Duplicate fermentations were performed using intracellular expressionstrains CLD977 and CLD990, and periplasmic strains CLD981 and CLD982.Specifically, the fermentation analysis was carried out in 3 stages:Shake flask (SF) seed stage, Fermenter stage, and SDS-PAGE analysisstage. To carry out the SF seed stage, RCB vials were inoculated intoduplicate shake flasks with standard media and incubated at 37° C., 200rpm for approximately 10 hours. Next, purity and OD₆₀₀ of the sampleswas determined (summarized in Table 1). Finally, the E. coli materialwas transferred from SF to a fermentation vessel.

TABLE 1 Results from the shake flask seed stage for intracellularstrains CLD977 and CLD990. SF1 and SF2 correspond to duplicate reactionsfor CLD977 and CLD990, respectively. Incubation Final OD600 Transfer SFRCB vial time OD600 Purity values to SF1 CLD977 9.97 h 3.60 Pure 3.60NBJ1605-04 SF2 B3214 9.97 h 3.61 Pure 3.61 C3 (P3A) SF1 CLD990 9.97 h1.37 Pure 1.37 NBJ1605-07 SF2 B3214 9.97 h 1.55 Pure 1.55 C4 (P3A)

The fermenter stage was conducted using the standard pAVEway™intracellular protocol. Specifically, cultures were induced using 0.5 mMIPTG when OD₆₀₀=50±5. After induction, fermentation continued for anadditional 12 hours before shutdown. Purity of the samples was confirmedat both pre-induction and shutdown.

For CLD977, the fermentation control parameter steps were: i) Oxygensupplementation at 7.33 hours; ii) End of batch phase at 9.46 hours whenfeed started; iii) Induction at 10.27 hours when OD₆₀₀=50.1; iv)Fermentation continued for a further 12 hours before shutdown.

As shown in FIG. 1 (a multi-fermenter computer system (MFCS) plot ofCLD977 fermentation), at approximately 20 hours, the airflow began tofail, which was suspected to be due to pressure in the vessel. Also,shown in FIG. 1, pO2 fell below 20% at approximately 21 hours and 1.5hours prior to shut down.

For CLD990, the fermentation control parameter steps were: i) Oxygensupplementation at 10.95 hours; ii) End of batch phase at 12.27 hourswhen feed started; iii) Induction at 13.14 hours when OD₆₀₀=50.1; iv)Fermentation continued for a further 12 hours before shutdown.

A MFCS plot of CLD990 fermentation is shown in FIG. 2.

A MFCS plot of exit gas analysis of oxygen uptake rate (OUR) and carbondioxide evolution rate (CER) for CLD977 and CLD990 fermentation is shownin FIG. 3. Similar profiles were observed for both strains with thedelay seen on the CLD990 strain due to an observed longer batch phase.Profile at the end of CLD977 fermentation, without wishing to be boundby theory, was probably related to a reduced airflow in the vessel (exitfilter blocked).

Biomass profiles for both strains were similar up to 12 hours postinduction although the CLD990 strain was delayed due to the extendedbatch phase (see FIG. 4). This delay, without wishing to be bound bytheory, may have been due to the lower SF OD₆₀₀ or a reduced initialgrowth rate.

Gram staining was also performed for CLD977 and CLD990 at the end ofbatch phase and after fermentation was complete (see FIG. 5). Resultsindicate that the culture was pure and homogenous at the end of theculturing.

Following fermentation, selected time course samples from pre-inductionto the end of fermentation were analyzed using SDS-PAGE (see FIGS. 6-8)after samples were lysed, spun down, and resuspended.

As evidenced by the SDS-PAGE results, protein product levels in bothstrains were in excess of 10 g/L (see FIGS. 6 and 7): CLD977 SDS PAGEindicated 12.1-14.0 g/L whereas CLD990 SDS PAGE indicated 13.2-13.7 g/L.Additionally, the CLD977 and CLD990 total protein products (aftersonication) were mostly soluble (see FIG. 8). Compared to previoussystems used to express β-lactamase (that yielded about 0.5 to 1 g/L),the methods of the present invention utilizing intracellular expressionof β-lactamase in E. coli strains proved to be far superior. Contrary toprior studies which show periplasmic β-lactamase expression, attempts toexpress β-lactamase in the periplasmic domain were unsuccessful and ledto biologically inactive β-lactamase (see Example 3).

Example 4: β-Lactamase Activity of Fermentation Samples by the CENTAMethod

P3A β-lactamase activity of the previously described fermentationsamples (see Example 2) was analyzed using the CENTA method, which isdescribed below. Throughout this method, different standards were usedand are referred to as: Reference material (32 mg/mL); Standard curve:Reference standard material diluted ×1000 (standards used were 0.6 mg/l,0.8 mg/l, 1.0 mg/l, 1.5 mg/l, 2.0 mg/l and 4.0 mg/l made up from the×1000 stock); Control standard: Reference standard diluted to 1 mg/l andran as a control; 1 mM CENTA stock solution: 25 mg CENTA lactamasesubstrate dissolved in 50 ml of 50 mM Sodium dihydrogen phosphate(stored at −20° C.); and CENTA working solution: 3.34 ml of CENTA stocksolution dissolved in 25 ml of Sodium dihydrogen phosphate.

The CENTA method employs a chromogenic cephalosporin that is readilyhydrolyzed by β-lactamases and allows for kinetic studies and detectionof the enzymes in crude extracts and chromatographic fractions (Bebrone,C. et al., (2001) Antimicrobial Agents and Chemotherapy, 45 (6)1868-1871). This method is also useful since CENTA can be prepared fromthe commercially available drug, cephalothin. For this study,β-lactamase sample activity was monitored using a FFDB plate reader inthe presence of a CENTA working solution. First, β-lactamase sampleswere diluted to 1 mg/l (Bradford assays were used to determine theconcentration). Then, 50 μL of each sample was loaded onto the plate andincubated for 20 min. at 25° C. Finally, 200 μL of the CENTA workingsolution was added to each sample and the sample was read as follows:Plate reader settings: Temperature of measurement=25° C.; Shaking=slow;Time of shaking=2 seconds; Time of measurement=60 seconds; Number ofreadings=Every 3 seconds; and Wavelength=405 nm.

The hydrolysis of CENTA was monitored by continuously recording theabsorbance variation at 405 nm (appearance of the expulsed chromophore).Results from this assay are presented in FIGS. 9-19 and summarized inTables 2-4.

The CENTA experiments were split into 3 assay plates. Assay plate 1corresponded to: CLD981 12 h, 24 h, 48 h, osmotic shock buffer 1 (OS1)24 h, and osmotic shock buffer 2 (OS2) 24 h post induction, as well asCLD982 12 h, 24 h, 48 h, OS1 24 h, and OS2 24 h post induction. Assayplate 2 corresponded to: CLD981 OS1 48 h and OS2 48 h post induction, aswell as CLD982 OS1 48 h and OS2 48 h post induction. Assay plate 3corresponded to CLD977 and CLD990 for both the second to last and lasttime point post induction (sonication) as well as the last time pointpost induction (Bug buster). OS1 contains 20% sucrose. Followingpreparation of the OS1 fraction, the cell pellet went on to preparationof OS2, which contains MgSO₄.

Results for assay plate 1 results are shown in FIGS. 9-13 and Table 2.Specifically, FIG. 9 shows a standard curve of Time (sec) vs. Absorbancefor Controls 1 and 2 (combined into control standard) as well asreference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4mg/L. Controls 1 and 2 were preset dilutions of 1.0 μg/mL ran asduplicates. FIG. 10 shows a standard end point curve of StandardConcentration (mg/L) vs. End Point Absorbance. Standard absorbance wasmeasured at time=60 sec minus standard absorbance at time=0 sec.Specifically, endpoint analysis was carried out in which a reaction wasmeasured at t=0 and at the end of a specified time interval, and the t=0absorbance value was subtracted. For analysis of beta-lactamase, thereaction was measured at time=60 sec. The absorbance was measured attime=0 sec which was then subtracted from the 60 sec measurement.Several dilutions of the reference standard were tested to generate astandard curve. FIG. 11 shows a standard curve of Time (sec) vs.Absorbance for CLD981 (3/13C037) 12 h, 24 h, 48 h, and OS2 48 h postinduction. FIG. 12 shows a standard end point curve of Time (sec) vs.Absorbance for CLD981 OS1 samples. FIG. 13 shows a standard curve ofTime (sec) vs. Absorbance for CLD982 (4/13C038) 12 h, 24 h, 48 h, andOS1 and OS2 48 h post induction. Table 2 shows a summary of assay plate1 activity and titre results for CLD981 and CLD982 (secretion strains 37and 38, respectively) along with controls 1 and 2.

Assay plate 2 results are shown in FIGS. 14-16 and Table 3.Specifically, FIG. 14 shows a standard curve of Time (sec) vs.Absorbance for Control 1 and 2 (combined into control standard) as wellas reference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and4 mg/L. FIG. 15 shows a standard end point curve of StandardConcentration (mg/L) vs. End Point Absorbance. Standard absorbance wasmeasured at time=60 sec minus standard absorbance at time=0 sec. FIG. 16shows a standard curve of Time (sec) vs. Absorbance for CLD981 (37) andCLD982 (38) OS1 and OS2 48 h post induction. Table 3 shows a summary ofassay plate 2 activity and titre results for CLD981 and CLD982 OS1 andOS2 along with controls 1 and 2.

Assay plate 3 results are shown in FIGS. 17-19 and Table 4.Specifically, FIG. 17 shows a standard curve of Time (sec) vs.Absorbance for Control 1 and 2 (combined as control standard) as well asreference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4mg/L. FIG. 18 shows a standard end point curve of Standard Concentration(mg/L) vs. End Point Absorbance. Standard absorbance was measured attime=60 sec minus standard absorbance at time=0 sec. FIG. 19 shows astandard curve of Time (sec) vs. Absorbance for CLD977 and CLD 990(intracellular strains 39 and 40, respectively) for both the second tolast and last time point post induction (unlabelled=sonication) as wellas the last time point post induction (Bug buster). Table 4 shows asummary of assay plate 3 activity and titre results for CLD977 andCLD990 along with controls 1 and 2.

Tables 2-4 specifically show CLD981, CLD982, CLD977, and CLD990 endpoint OD, activity concentration (mg/L), assay dilution,concentration×dilution (g/L), whole cell weight (WCW (g/L)), periplasmicdilution factor, g/L P1A activity WB titre, estimated g/L P1A WB by SDSPAGE, SDS PAGE P, and SDS PAGE soluble (if applicable) compared tocontrol 1 and 2.

TABLE 2 Results from the CENTA method for the periplasmic strains CLD981(37) and CLD982 (38) at different time points compared to controls 1 and2. Estimate d g/L P1A End Activity Concentration × Periplasmic g/L P1AWB by Point concentration Assay dilution dilution activity SDS Sample OD(mg/L) dilution (g/L) WCW g/L factor WB titre PAGE 37 12 hrs SN 0.0090−0.0710 217 0.00 119.0 0 >0 37 24 hrs SN 0.0155 0.0010 357 0.00 122.80.00 >0 37 48 hrs SN 0.0570 0.4608 714 0.33 161.4 0.28 0.05 37 OS1 24 h0.5195 5.5850 250 1.40 0.862 1.62 >0 37 OS2 24 h 0.0420 0.2946 1250 0.370.862 0.43 0.8 38 12 hrs SN 0.0270 0.1285 357 0.05 123.9 0.04 >0 38 24hrs SN 0.1090 1.0369 2083 2.16 125.1 1.89 0.35 38 48 hrs SN 0.11501.1034 5155 5.69 122.1 4.99 0.8 38 OS1 24 h 0.1025 0.9649 4587 4.43 1.064.18 0.8 38 OS2 24 h 0.1440 1.4247 1613 2.30 10.6 2.17 0.4 Assay control1 0.1210 1.1699 32000 37.44 Assay control 2 0.1245 1.2087 32000 38.68

TABLE 3 Results from the CENTA method for the periplasmic strains CLD981(37) and CLD982 (38) at different time points compared to controls 1 and2. Activity Concentration × Periplasmic g/L P1A SDS End Pointconcentration Assay dilution dilution activity WB PAGE Sample OD (mg/L)dilution (g/L) factor titre g/L WB 37-14 OS1 0.0420 0.2946 417 0.12 0.710.17 0.05 37-14 OS2 0.0270 0.1285 1786 0.23 0.71 0.32 1.3 38-14 OS10.1090 1.0369 2294 2.38 1.44 1.65 0.4 38-14 OS2 0.1150 1.1034 1250 1.381.44 0.96 0.3 Assay control 1 0.1140 0.9730 32000 31.14 Assay control 20.1145 0.9780 32000 31.30

TABLE 4 Results from the CENTA method for the intracellular strainsCLD977 (39) and CLD990 (40) at different time points compared tocontrols 1 and 2. Activity Activity concentration × g/L P1A SDS EndPoint concentration Assay dilution activity SDS PAGE Sample OD (mg/L)dilution (g/L) WB titre PAGE P soluble 39-7 (Sonication) 0.1980 1.970520830 41.05 41.05 11.4 10.9 39-8 (Sonication) 0.1875 1.8593 25000 46.4846.48 14.0 12.1 40-7 (Sonication) 0.1835 1.8169 25000 45.42 45.42 11.511.8 40-8 (Sonication) 0.1865 1.8487 27780 51.36 51.36 13.2 13.7 39-8(Bug buster) 0.1820 1.8010 25000 45.03 45.03 14.0 12.1 40-8 (Bug buster)0.1770 1.7480 27780 48.56 48.56 13.2 13.7 Assay control 1 0.1060 0.995732000 31.86 Assay control 2 0.1040 0.9745 32000 31.18

As seen above, for the intracellular strains, there was a marginallygreater activity in strain CLD990 compared to strain CLD977. For theperiplasmic strains, the best secretion fraction for CLD981 (gene 1leader) was OS2 fraction at 1.3 g/L (by SDS-PAGE), whereas the bestsecretion fraction for CLD982 (gene 7 leader) was SN fraction at ˜1.0g/L (which included the non-processed form). Finally, for intracellularstrains, it was observed that applying either Bug buster or sonicationproduced similar activity and SDS-PAGE results for these preparations.

Overall, intracellular activity and SDS PAGE results were more than 10×greater compared to secretion (periplasmic) fractions. This was asurprising result as typically, expressed proteins are collected fromthe periplasm. Additionally, the intracellular expression yieldedβ-lactamase in the soluble fraction as opposed to inclusion bodies.

Example 5: Large Scale P3A (SYN-004) Production

cGMP manufacturing of P3A (SYN-004) was undertaken. The initial750-liter cGMP production run used the pAVEway™ platform (FUJIFILMDiosynth Biotechnologies UK). Yields were 5.5 kilograms of >95% pureSYN-004 active pharmaceutical ingredient (API) drug substance. The GMPmanufacturing process was initiated after a successful evaluation thatproduced high yielding cell lines that exhibited consistent biologicalactivity of P3A (SYN-004). P3A (SYN-004) expression titers were improvedby greater than about 15-fold (14 grams of P3A (SYN-004) per liter of E.coli culture broth), compared to the Bacillus platform previouslyemployed for SYN-004's first-generation predecessor (roughly 1 gram ofHA per liter of Bacillus subtillis culture broth, see Example 1). Asingle chromatography column purification process reproducibly yielded40-50% P3A (SYN-004) drug substance recovery at purity levels greaterthan 95%, another marked manufacturing improvement over the previouspurification process.

Definitions

The following definitions are used in connection with the inventiondisclosed herein. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofskill in the art to which this invention belongs.

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disease ofinterest.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

The amount of compositions described herein needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering therapeutic agents (e.g. inventive β-lactamases and/orpharmaceutical compositions (and/or additional agents) for therapeuticpurposes, the therapeutic agents are given at a pharmacologicallyeffective dose. A “pharmacologically effective amount,”“pharmacologically effective dose,” “therapeutically effective amount,”or “effective amount” refers to an amount sufficient to produce thedesired physiological effect or amount capable of achieving the desiredresult, particularly for treating the disorder or disease. An effectiveamount as used herein would include an amount sufficient to, forexample, delay the development of a symptom of the disorder or disease,alter the course of a symptom of the disorder or disease (e.g., slow theprogression of a symptom of the disease), reduce or eliminate one ormore symptoms or manifestations of the disorder or disease, and reversea symptom of a disorder or disease. For example, administration oftherapeutic agents to a patient suffering from a GI tract disorder (e.g.ODD provides a therapeutic benefit not only when the underlyingcondition is eradicated or ameliorated, but also when the patientreports a decrease in the severity or duration of the symptomsassociated with the disease. Therapeutic benefit also includes haltingor slowing the progression of the underlying disease or disorder,regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to about 50% ofthe population) and the ED50 (the dose therapeutically effective inabout 50% of the population). The dosage can vary depending upon thedosage form employed and the route of administration utilized. The doseratio between toxic and therapeutic effects is the therapeutic index andcan be expressed as the ratio LD50/ED50. In some embodiments,compositions and methods that exhibit large therapeutic indices arepreferred. A therapeutically effective dose can be estimated initiallyfrom in vitro assays, including, for example, cell culture assays. Also,a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the 1050 as determined in cellculture, or in an appropriate animal model. Levels of the describedcompositions in plasma can be measured, for example, by high performanceliquid chromatography. The effects of any particular dosage can bemonitored by a suitable bioassay. The dosage can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment.

In certain embodiments, the effect will result in a quantifiable changeof at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 70%, or at least about 90%. In someembodiments, the effect will result in a quantifiable change of about10%, about 20%, about 30%, about 50%, about 70%, or even about 90% ormore. In certain embodiments, the effect will result in a quantifiablechange of two-fold, or three-fold, or four-fold, or five-fold, orten-fold. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder or reduction intoxicity, regardless of whether improvement is realized.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein set forth and as follows in the scope of theappended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

What is claimed is:
 1. A method for the production of a beta-lactamasepolypeptide in Escherichia coli (E. coli), comprising: (a) providing ahost E. coli cell transformed with an expression vector comprising asequence encoding the beta-lactamase polypeptide; (b) culturing the E.coli cell to induce expression of the beta-lactamase polypeptide in thecytoplasm; and (c) recovering the beta-lactamase polypeptide from acytoplasmic soluble fraction prepared from the E. coli cell; and whereinthe method yields more than 10 grams of the beta-lactamase polypeptideper liter of culture, wherein the beta-lactamase polypeptide comprisesan amino acid sequence having at least 95% identity with a sequenceselected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQID NO: 7 and SEQ ID NO: 10, wherein the host E. coli cell is selectedfrom BL21 (DE3) or W3110, and wherein the beta-lactamase polypeptide isnot recovered or purified from a periplasmic fraction, a cell envelopefraction, a cytoplasmic insoluble fraction, an inclusion body, or anextracellular fraction of the E. coli cell.
 2. The method of claim 1,wherein the method yields more than 15 grams of the beta-lactamasepolypeptide per liter of culture.
 3. The method of claim 1, whereinexpression of the beta-lactamase polypeptide in the cytoplasm is inducedby adding isopropylthiogalactoside (IPTG) to the culture.
 4. The methodof claim 1, wherein the beta-lactamase polypeptide comprises the aminoacid sequence of SEQ ID NO:
 5. 5. The method of claim 1, wherein thebeta-lactamase polypeptide comprises the amino acid sequence of SEQ IDNO: 10 (P2A).
 6. The method of claim 1, wherein the expression vectorcomprises palindromic DNA looping.
 7. The method of claim 1, wherein theexpression vector is capable of tightly controlled gene expression.
 8. Amethod for the production of a beta-lactamase polypeptide in Escherichiacoli (E. coli), comprising: (a) providing a host E. coli celltransformed with an expression vector comprising a sequence encoding thebeta-lactamase polypeptide; (b) culturing the E. coli cell to induceexpression of the beta-lactamase polypeptide in the cytoplasm; and (c)recovering the beta-lactamase polypeptide from a cytoplasmic solublefraction prepared from the E. coli cell; and wherein the beta-lactamasepolypeptide comprises an amino sequence selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO:10 and the method yields more than 15 grams of the beta-lactamasepolypeptide per liter of culture, wherein the host E. coli cell isselected from BL21 (DE3) or W3110, and wherein the beta-lactamasepolypeptide is not recovered or purified from a periplasmic fraction, acell envelope fraction, a cytoplasmic insoluble fraction, an inclusionbody, or an extracellular fraction of the E. coli cell.